A rising need for workers in science, technology, engineering and mathematics (STEM) fields has fueled interest in improving teaching within STEM disciplines. Numerous studies have demonstrated the benefits of active learning approaches on student learning outcomes. However, many of these studies have been conducted in experimental, rather than real-life class, settings. In addition, most of these studies have focused on in-class active learning exercises. This study tested the effects of answering questions outside of class on exam performance for General Biology students at the University of Minnesota. An online database of 1,020 multiple-choice questions covering material from the first half of the course was generated. Students in seven course sections (with an average of ∼265 students per section) were given unlimited access to the online study questions. These students made extensive use of the online questions, with students answering an average of 1,323 questions covering material from the half of the semester for which the questions were available. After students answered a set of questions, they were shown the correct answers for those questions. More specific feedback describing how to arrive at the correct answer was provided for the 73% of the questions for which the correct answers were not deemed to be self-explanatory. The extent to which access to the online study questions improved student learning outcomes was assessed by comparing the performance on exam questions of students in the seven course sections with access to the online study questions with the performance of students in course sections without access to the online study questions. Student performance was analyzed for a total of 89 different exams questions that were not included in the study questions, but that covered the same material covered by the study questions. Each of these 89 questions was used on one to five exams given to students in course sections that had access to the online study questions and on three to 77 exams given to students in sections that lacked such access. Data from over 1,800 students in sections with access to the online study questions show that those students scored a statistically significant average of 6.6% points higher on the exam questions analyzed than students in sections without access to the study questions. This difference was greater than the average amount necessary to raise students’ exam grades by one grade . In addition, there was a higher correlation between number of questions answered and success on exam questions on material related to the study questions than between number of questions answered and success on exam questions on material unrelated to the study questions. The online study question system required substantial effort to set up, but required minimal effort to maintain and was effective in significantly raising average exam scores for even very large course sections. Due to a rising need for highly trained workers in science, technology, engineering and mathematics (STEM) fields , and forAlthough intriguing, these studies leave open the question of the extent to which providing students with access to study questions can improve overall student performance in a real classroom. To address this question, students in seven sections of BIOL 1009 General Biology at the University of Minnesota Twin Cities were provided with unlimited access to an online study question database. This online database contains 1,020 multiple-choice questions on material taught during the first half (14 lectures) of the course. After students answered a set of questions, they were shown the correct answers for those questions. More specific feedback describing how to arrive at the correct answers was provided for the 73% of the questions for which the correct answers were not deemed to be self-explanatory. The effects of access to the online study questions were assessed by comparing the performance of students with access to the online study questions to the performance of students without access to the online study questions on in-class exam questions. Use of the online study questions was found to significantly improve student performance on exams.BIOL 1009 General Biology is a one-semester (14–15 week) course. It is taught in multiple sections each term on the University of Minnesota Twin Cities campus. This course is intended for students who are not within the College of Biological Sciences, but who do have a specific need to learn biology . Course sections meet twice per week for 75 min each time. Course sections meet an additional time each week for a 2-h laboratory session. The course as a whole (lecture plus lab) is a four-credit course. Most course instructors, including the author, teach the course using primarily a traditional lecture format, but include some in-class active learning exercises as well. These in-class active learning exercises may include answering a few questions, think-pair-share or similar activities. All instructors for BIOL 1009 use the same textbook (Campbell Biology). The author taught the first half (lectures one through 14) of BIOL 1009 section 30 in 2012 and sections 1 and 30 in 2013–2015. The sections taught by the author had approximately 220 to 320 students per section.https://moodle.org/). The question bank contains a total of 1,020 questions covering material taught by the author during the first 14 lectures  of BIOL 1009 General Biology. There are an average of 73 questions per lecture, with a range of 47–98. All of the questions in the question bank are multiple-choice questions. Each question has a single “best” answer for which full credit is assigned and three distractors for which no credit is assigned. Most of the questions fit into one of the three lower levels  of a revised version (http://www.pearsonmylabandmastering.com/northamerica/masteringbiology/) that cover material from Campbell Biology Ninth Edition  in answering quiz questions. Students were allowed to attempt each quiz a maximum of three times, with only their highest score for each quiz being counted. In addition, the lowest two quiz scores were dropped for each student. These quizzes, as a whole, accounted for 10% of students’ course grades for the first half of the semester, or 5% for the course as a whole.The Biology Program at the University of Minnesota Twin Cities established a database of exam questions for BIOL 1009 General Biology in fall 1978. Each term new questions are added to this database by the different faculty members teaching the course. This database currently contains 2,284 active questions covering material from the entire course. Records are maintained for each question. These records indicate each course section for which that question was used on an exam and the percentage of students that answered the question correctly each time it was used on an exam. Using this information, an “expected” exam score can be calculated for each group of questions chosen from the exam question database. These “expected” exam scores are calculated by averaging the average facility indexes for the chosen questions. For this work, “expected” scores were calculated using only results from questions that had been used at least three times in exams for course sections for which online study questions were not available, and that were not honors sections. There were a total of 89 such questions. On average, the questions from the exam question database analyzed as part of this work were used in 24.8 exams for course sections for which online study questions were not available and that were not honors sections . In totaThere were three course exams. All exams took place in class and lasted for a maximum of 75 min (exams 1 and 2) or 120 min . Students were not allowed to use any resources  during exams. All exams consisted of multiple-choice questions with one “best” answer and three distractors. There were 40 questions each for exams 1 and 2 and 50 questions for the final exam. Exams 1 and 2 each accounted for 16.67% of students’ final course grades and the final exam accounted for 20.83% of final course grades. Exam 1 covered material from lectures one through eight. Questions for exam 1 were drawn from two sources. For each exam 1, 20–21 questions were chosen by the author from the online study questions. The remaining 19–20 questions  were choThe University of Minnesota Twin Cities IRB: Human Subjects Committee determined that the referenced study (study number 1508E77489) is exempt from review under federal guidelines 45 CFR Part 46.101(b) category #4 EXISTING DATA; RECORDS REVIEW; PATHOLOGICAL SPECIMENS. The author received this exempt study notification from the IRB via email.t-test. Correlations between numbers of questions answered versus performance on different groups of exam 2 questions were calculated as the Pearson product moment correlation coefficient, r. Calculations of the p-values for different r values were performed using an online calculator .“Actual” versus “expected” scores for questions chosen from the Biology Program exam question database were analyzed using a two-tailed, paired Student’s A database of 1,020 questions covering material from the first half (first 14 lectures) of BIOL 1009 General Biology was developed in 2012. These questions were made available online to students in seven course sections from 2012 to 2015. Students answered questions from this database both as sets of optional online “study questions” and as mandatory online quizzes. Immediately after answering a set of study questions or a quiz, students were shown the correct answers for each question. Questions they had answered incorrectly were also indicated. More detailed feedback explaining how to arrive at the correct answer or why a particular distractor is incorrect was provided for 73% of the questions. The answers for the remaining 27% of the questions were self-explanatory. This form of delayed feedback  has been shown to be particularly effective in promoting learning . A conceFrom 2012 to 2015, students answered an average of approximately 1,323 questions (an average of 94.5 questions per lecture) for the 14 lectures for which online questions were available . As a reThe majority of the questions answered were optional study questions for which no credit was available, rather than quiz questions for which credit towards final course grades was available. In 2012, for example, 76% of the questions answered by students were study questions . The aveThe Biology Program at the University of Minnesota maintains a database of questions that have been used on BIOL 1009 General Biology exams. Records are kept for each question in the exam question database. These records indicate the exams on which the question was used and the fraction of students who answered the question correctly (the facility index) on each exam. Using this information, it is possible to calculate an expected exam score for questions chosen from this exam question database. For the seven sections of BIOL 1009 that are the focus of this study, 19–20 questions from the Biology Program database were chosen for exam 1 each year and 10–13 questions were chosen for exam 2 each year. The author used the same exams for both sections of the course taught in the same year. In addition, some questions were used on exams in more than one year. To help ensure the accuracy of the results, only questions that had been used by a minimum of three course sections for which online study questions were not available (and that were not honors sections of the course) were analyzed. Consequently, a total of 89 questions (50 exam 1 and 39 exam 2 questions) chosen from the Biology Program exam question database were analyzed. These questions were used on an average of 24.8 exams, with a range of 3–77 exams, for courses for which study questions were not available .The actual scores achieved by students in the seven course sections for which online study questions were available were consistently higher than expected . The comr) of 0.36 . This study therefore indicates the desirability of providing students with opportunities for more active forms of learning outside of class. The online study question system analyzed in this work provides an effective means of achieving this goal. It requires substantial effort to set up in the first year, but requires minimal effort to maintain in subsequent years. In addition, it is effective in significantly raising average exam scores for even very large course sections.10.7717/peerj.1322/supp-1Table S1This table summarizes information on the exam questions from the University of Minnesota Biology Program exam question database for BIOL 1009 that were analyzed as part of this work. “Mean actual score” refers to the percentage of students who correctly answered the indicated question on the indicated exam for the indicated year and course section. “Mean expected score” refers to the percentage of students who correctly answered the indicated question on all exams in which the question was used, excluding exams from one of the seven course sections being analyzed as part of this work  and from honor’s sections. ”# of other exams on which question was used” refers to the number of times the indicated question has been used on a BIOL 1009 exam at the University of Minnesota, excluding exams from one of the seven course sections being analyzed as part of this work and exams from honor’s sections of BIOL 1009.Click here for additional data file.10.7717/peerj.1322/supp-2Table S2The numbers of study questions, quiz questions and total questions (study plus quiz questions) answered by each student in preparation for exams 1 and 2 during 2012 are shown. The overall exam 1 and 2 scores for each student are also indicated. In addition, the scores on different groups of exam 2 questions are shown. “Exam 2 study questions” refers to questions on exam 2 that were taken directly from the online study questions. “Questions on same material” refers to questions covering the same material covered by the online study questions; these questions were chosen by the author from the exam question database maintained by the Biology Program at the University of Minnesota (thus these questions were not written by the author). “Questions on other material” refer to questions on material not covered by the study questions; these questions were chosen by a different faculty member.Click here for additional data file.10.7717/peerj.1322/supp-3Table S3The total numbers of questions (study questions plus quiz questions) answered by each student in preparation for exams 1 and 2 in 2012 and 2013 are indicated. The average number of questions answered per lecture for each student in preparation for exams 1 and 2 are also shown. The questions in preparation for exam 1 covered material from eight lectures and the questions in preparation for exam 2 covered material from six lectures.Click here for additional data file.10.7717/peerj.1322/supp-4Table S4In 2012 through 2014, questions for exam 2 were drawn from three sources. 13–15 questions were chosen by the author from the online study questions, 11–13 questions that covered the same course material covered by the online study questions were chosen by the author from questions in the exam question database maintained by the Biology Program at the University of Minnesota  and 13–16 questions were chosen by another faculty member and covered material taught by that faculty member that was not covered by the online study questions . The number of questions answered by each student in preparation for exam 2 and the scores obtained by that student on the three groups of exam 2 questions and exam 2 overall are indicated.Click here for additional data file.


Interestingly, RCMGO-24 demonstrated a higher specific capacitance and excellent cycle stability due to its residual oxygen functional groups that accelerate the faradaic reactions and aid in faster wetting. This non-annealed strategy offers the potential for simple and cost-effective preparation of an active material for a supercapacitor electrode.An efficient active material for supercapacitor electrodes is prepared by reacting potassium hydroxide (KOH) with graphene oxide followed by chemical reduction with hydrazine. The electrochemical performance of KOH treated graphene oxide reduced for 24 h  exhibits a specific capacitance of 253 F g Supercapacitors or electrochemical capacitors, powerful energy storage systems used for a wide range of energy capture and storage applications, provide clean energy with almost zero waste emission -3. HowevGraphene can be prepared by the chemical reduction of graphene oxide (GO) -18, epitHerein we report on the preparation, through simple reactions of KOH with graphene oxide followed by hydrazine reduction, of an active material of graphene with adequate dispersibility and high storage capacity. The reactions of KOH induce the formation of oxygen functional groups and defects on graphene sheets. The reduced KOH-treated graphene oxide was analyzed with thermal gravimetric analysis (TGA), Raman, Fourier transform infrared spectroscopy (FT-IR), and electrochemical charge/discharge cycles to elucidate the physical and electrochemical performances. KOH-treated reduced graphene oxide exhibits adequate dispersibility in an aqueous medium; consequently, it induces good wettability in an aqueous electrolyte solution. Due to the presence of residual oxygen functional groups for pseudocapacitance in this KOH-treated graphene oxide, higher storage capacitance and good electrochemical stability is achieved.2SO4), potassium permanganate (KMnO4), hydrochloric acid (HCl), and hydrogen peroxide (H2O2) were purchased from Samchun Chemical . Hydrazine monohydrate (N2H4∙H2O) and KOH was purchased from Sigma-Aldrich . All chemicals were used as received without further purification.Expandable graphite  was kindly provided by Asbury Carbon. Concentrated sulfuric acid  was added and stirred for each reaction time. The reduced GO was filtered and washed with water, then dried in an oven at 80°C for 24 h. The reduced CMGO (RCMGO) sample was also prepared by applying same reaction conditions of RGO to CMGO. The sample of GO reduced for 6 h, 24 h without KOH treatment, and CMGO reduced for 24 h were named RGO-6, RGO-24, and RCMGO-24, respectively.-1. Cyclic voltammetry (CV) was conducted at 10 mV s-1 and electrochemical impedance spectra (EIS) was measured at frequencies from 100 kHz to 0.01 Hz.All electrochemical measurements were conducted in a three-electrode cell assembly equipped with a reference electrode (Ag/AgCl), platinum wire as a counter electrode, and a glassy carbon working electrode. To fabricate the electrode, 85% active material, 10% super P, and 5% polytetrafluoroethylene were mixed with ethanol and then dried to form a paste. Electrochemical performance was measured in a 2 M aqueous sulfuric acid solution as an electrolyte. The specific capacitances of RGO-6, RGO-24, and RCMGO-24 were measured at 0.2, 0.5, and 1 A gθ range from 2° to 40°. X-ray photoelectron spectroscopy (XPS) was taken on a Thermo Fisher instrument using Al Kα radiation (energy range 200 to 3 keV). Galvanostatic charge/discharge and CV were characterized using a Won A Tech (WBCS 3000) battery tester. EIS was measured using BioLogic Science Instruments .Morphologies of the RGO-24 and RCMGO-24 were characterized via field emission scanning electron microscopy . Thermal properties were characterized using thermal gravimetric analysis . The electrical conductivity of the graphene pellet was determined by the four-point probe method . The functional groups of the RGO were determined by FT-IR on a Nicolet IR 200 FT-IR spectrometer  in transmission mode, and Raman  with 633-nm wavelength incident laser light was used to characterize the degree of reduction on the graphene sheets. X-ray diffraction (XRD) analyses were performed on a high-power X-ray diffractometer  at 2As shown in Figure -1. The electrical conductivity of RGO-24 was measured to be as high as 1,980 S m-1. On the other hand, the residual functional groups obtained from KOH reaction diminish electron transport of RCMGO-24 and thus the electrical conductivity decreases to 431 S m-1. Nevertheless, there is enough electrical conductivity for charge transport during charge/discharge cycles.TGA was carried out to observe the presence of functional groups on the graphene sheets. As shown in Figure -1 originates from the stretching vibration of OH groups, and a peak at 1,727 cm-1 is due to the C = O stretching vibrations of carbonyl and carboxyl groups. The C = C of an un-oxidized graphitic domain gives a skeletal vibration at 1,590 cm-1. The peaks at 1,230 and 1,050 cm-1 are characteristics of carboxyl C-O deformation and alkoxy C-O stretching vibrations, respectively. As seen in the figure, the peak intensities of RGO-24 and RCMGO-24 were lower than those of GO, suggesting effective removal of functional groups by hydrazine. However, these graphene samples still have some surface functional groups over the basal and edge planes of the graphene sheets, which were observed at 3,400, 1,560, and 1,205 cm-1 for residual -OH, C = C, and C-O groups, respectively. The broad peak observed at 1,205 cm-1 for RCMGO-24 denotes the greater presence of residual oxygen functional groups.As shown in FT-IR spectra Figure b the bro3/2 and K2p1/2 peaks at 292.9 and 295.5 eV, respectively  yielded a value very near to that of GO. Interestingly, the reactions of CMGO at higher temperature accelerated the formation of new oxygen functional groups, so that the D band of RCMGO-24 was observed at a similar level to that of GO. Moreover, the degree of reduction and defects were also determined by calculating the ID/IG ratio of GO, RCMGO-24, and RGO-24. The ID/IG ratio of GO was calculated to be 0.97. After the reduction, the D band of GO increased to more than that of the G band (ID/IG = 1.4), suggesting that the reaction of hydrazine formed a higher reduction and larger number of defects over the graphene sheets [ID/IG ratio of RCMGO-24 is much smaller than that of RGO-24. The ID/IG ratio of RCMGO-24 was calculated to be 1.15; this small number results from the formation of a large number of oxygen functional groups on the defected areas. From XRD analysis  restored its graphitic structure, and then formed a stacked, multilayer graphene.Raman spectroscopy was used to characterize the structures of graphene by comparing G and D bands. The G band corresponds to the Er number . For RGOe sheets ,29. It is Figure , compare-1 under a potential window of 0 to 1 V. The slope of a full discharge curve  drop) was chosen to measure the specific capacitance of RGO-24 and RCMGO-24. The specific capacitance of graphene was determined by using the following equation:Next, we performed the analysis in a supercapacitor electrode to evaluate the storage capacitance and cyclic stability of RGO-24 and RCMGO-24. We believe that the crumpled structure and residual oxygen functional groups obtained from KOH treatment can effectively increase the specific capacitance of graphene sheets. Galvanostatic charge/discharge was conducted at current densities of 0.2, 0.5, and 1 A gCspec is the specific capacitance of an electrode, I is the current density (A), m is the mass of the active material (g), and dv/dt is the slope of the discharge curve. Figure -1 measured after the 10 CV (10 mV s-1) cycles. The discharge curves are close to linear over the whole potential range, implying perfect capacitance behavior [-1. The maximum specific capacitances of RGO-24 and RCMGO-24 were observed at 141 and 253 F g-1at 0.2 A g-1, respectively. The specific capacitance of RCMGO-24 is comparable to the value reported for reduced graphene supercapacitors [where behavior . Secondlpacitors ,24. The -1  represents ESR and it corresponds to the internal charge, charge/discharge rate, and wettability of the electrode, [y-axis in comparison to RGO-24, thereby confirming the contribution of higher EDLC characteristics. These interesting characteristics of RCMGO-24 indicate that the KOH treatment decreased electrical conductivity, but it significantly increased the electrode wetting, electrolyte diffusion, EDLC, and more pseudocapacitance in comparison to RGO-24.Figure ectrode, ,32,33. TA non-annealing strategy of KOH-treated GO was developed for preparing RCMGO-24 containing residual functional groups. The reactions of KOH efficiently created the oxygen functional groups and were involved in the pseudocapacitance effect; the same characteristic features were observed using a solvothermal method at elevated temperature (150°C) . The cruWe have investigated the effect of KOH treatment of GO on supercapacitor performance at various current densities. The Raman, TGA, FT-IR, and XPS spectra of RCMGO-24 confirmed that KOH functionalization efficiently prevents complete reduction and supplies residual functional groups on graphene sheets. The excellent performance of supercapacitor electrodes obtained with RCMGO-24 illustrated the key role of the KOH treatment. It promoted faster wetting of the electrode, more pseudocapacitance, and the permeation of an electrolyte solution. The electrochemical stability of RCMGO-24 was sufficient and maintained capacitance retention for up to 3,000 cycles, demonstrating that it is an excellent active material for supercapacitor electrodes.CV: cyclic voltammetry; EIS: electrochemical impedance spectra; ESR: equivalent series resistance; EDLC: electrical double-layer capacitor; FT-IR: Fourier transform infrared spectroscopy; GO: graphene oxide; IR: internal resistance; RGO: reduced graphene oxide; RCMGO: reduced chemically modified graphene oxide; FE-SEM: field emission scanning electron microscopy; TGA: thermal gravimetric analysis; XRD: X-ray diffraction; XPS: X-ray photoelectron spectroscopy.The authors declare that they have no competing interests.BR designed the work, carried out the experiment, and finalized the manuscript. JSC supervised the work and revised the manuscript. All authors read and approved the final manuscript.BR is a PhD student at the University of Ulsan (UOU). JSC has a PhD degree and is a professor at UOU.


Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs.Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate  Genetic and epigenetic abnormalities have been found to result from reprogramming of differentiated cells into human induced pluripotent stem cells (hiPSCs). Here, Klawitter et al. identify endogenous L1, Alu and SVA mobilization during reprogramming, highlighting the risk of insertional mutagens in hiPSCs. Unlike human embryonic stem cells (hESCs), hiPSCs are a potential source of autologous cells compatible with the immune system of transplant recipientsin vitro2345Human induced pluripotent stem cells (hiPSCs) hold substantial promise for biomedical applications and as Alu and SVA retrotransposition, has caused widespread genome structural variation in human populations101213De novo retrotransposition events can profoundly alter gene structure, expression and function, and drive pathogenesis15168LINE-1 (L1) retrotransposons  are mobi910Alu and SVA mobilization in hiPSCs, and use an exemplar de novo L1 insertion in CADPS2 to demonstrate the potential impact of endogenous retrotransposition in pluripotent stem cells.Nonetheless, epigenome-wide remodelling20132023de novo in descendant hiPSCs. As additional controls, we used three hESC lines as benchmarks of L1 expression and pluripotency  using several combinations of reprogramming factors, as well as integrating and non-integrating delivery systems . Extensi2526ipotency .et al.P<0.05–P<0.0001, analysis of variance (ANOVA)), that peaked in earlier passages of cell lines hiPS-SB4 and hiPS-SB5  and resembled levels observed in hESCs . Consistently, L1 ORF1p was abundant in hiPSCs, based on immunoblot  to map the genomic integration sites of Alu, 53 SVA and 14 LTR non-reference genome insertions per hiPSC and hESC sample (de novo in pluripotent cells if they were not (i) reported previously in non-reference retrotransposon insertion databases9123334353637Alu and two SVA putative de novo insertions  known to be active in humans contributed putative de novo insertions1011Alu and one SVA insertion confirmed as de novo in hiPSCs and a single Alu insertion (Alu-2) in hESCs  were detected by PCR in parental cells or an earlier hESC passage, suggesting that these variants were present but were not de novo. Next, we determined the entire nucleotide sequence of 10/11 confirmed de novo retrotransposon insertions . Thus, we concluded that although RC-seq reliably detected high variant allele fraction retrotransposon insertions, a large pool of low variant allele fraction events may have been overlooked at the RC-seq thresholds used here. This would be particularly acute in the chosen hESC lines where, unlike iPSCs, cells had not undergone a recent population bottleneck in vitro. Using these parameters and the observed de novo L1 insertion counts, we estimated that hiPSC lines carried 3.7 de novo L1 insertions with allele frequencies ≥5%, on average, extrapolating to ∼1 de novo L1 insertion per cell (see Methods). However, the low number of insertions identified precluded similar estimates for hESC lines.The rate of L1-mediated retrotransposition occurring in pluripotent stem cells was difficult to accurately assess given the unknown genomic heterogeneity of each population. However, by estimating the sensitivity of RC-seq, we were able to determine the approximate L1 mobilization rate in hiPSCs. First, we identified that the overall RC-seq false positive rate was 1.5%, based on our recent PCR validation rate of 98.5% for insertions found by RC-seq in a cohort of hepatocellular carcinoma patientsng depth . At 50% de novo L1 insertions detected by RC-seq were absent from the earlier hiPSC passage surveyed and therefore may have arisen after reprogramming. To better establish the temporal dynamics of L1 retrotransposition in hiPSCs, we performed multiplex TaqMan qPCR incorporating a 5′ junction-spanning probe  of hiPS-SB4 isolated well before the later passages analysed by RC-seq (p43 and p53)  . L1-dn13and p53) . Hence, nsertion , can leade novo L1 insertions were full length, a surprising result given that most preexisting genomic L1 retrotransposition events are 5′ truncated3350de novo L1s  revealed no deleterious nonsense mutations in their ORFs  and nephron (SLC12A1) biology, as well as genes with established and predicted roles in cell cycle regulation and oncogenesis . Insertions showed a marked bias for the 5′ end of genes, with insertions falling on average in the 20th percentile of gene length measured from the annotated RefSeq transcription start site (TSS), a significant deviation from random expectation . Albeit based on a small sample of insertions, this outcome could be explained by L1 endonuclease preference for open chromatinSix CADPS2 and, as noted above, exhibited copy-number variation during hiPSC cultivation , (ii) 423 bp spanning the same region but in this case containing the 389 bp L1-dn13 insertion and its TSDs to produce a 825 bp sequence (pSHM06_02) and (iii) the 423 bp sequence on its own (pSHM06_03). We cloned each of these fragments into the triose phosphate isomerase/Renilla reporter cassette and quantified their effect on luciferase activity (CADPS2 intron sequence harbouring L1-dn13 (pSHM06_03) had the strongest inhibitory effect and reduced luciferase activity by 62%, a significant decrease beyond the constructs lacking L1-dn13 (P=0.022).To further test this possibility, we employed a human triose phosphate isomerase/Renilla luciferase reporter assay developed to monitor the effects of different introns on mammalian gene expressionactivity . InteresCADPS2 expression was ∼95% lower in hiPS-SB4_D than in hiPS-SB4_B  where L1-dn13 was identified by RC-seq, one carrying L1-dn13 (hiPS-SB4_D) and the other not carrying L1-dn13 (hiPS-SB4_B) . The idePS-SB4_B . ConsistPS-SB4_D , as distHere we have demonstrated that endogenous L1-mediated retrotransposition can occur in hiPSCs and hESCs, building upon earlier reports of engineered L1 retrotransposition in stem cells1320222333de novo L1 insertion, with the notable caveat that this calculation was based on a small number of observed events. Nonetheless, this is a much lower rate than recently found for human hippocampal neurons and glia de novo insertions with a variant allele fraction of <5% in hiPSC and hESC populations were overlooked by RC-seq at the detection thresholds used here, and these were not included in the above rate estimate. This is a major consideration in concluding whether parental cell type or choice of reprogramming vector affects endogenous retrotransposition activity in hiPSCs. Low frequency or subclonal retrotransposition may indeed occur in our hCBEC-derived hiPSC lines, that were reprogrammed via lentiviral systems, and escaped detection by RC-seq here. Therefore, we would propose that additional experiments are required to better define how these and other considerations  affect L1 activity. Indeed, one explanation for the low number of insertions characterized in hESCs is that these cell populations were not clonally derived and were therefore likely to present more extensive genomic heterogeneity than hiPSCs. The lone Alu insertion found here in H9 cells is nonetheless the first endogenous retrotransposition event reported in hESCs, reinforcing evidence that L1-mediated mobilization can occur in early human development13We estimated that hiPSCs each carried ∼1 per se may activate the expression of cellular L1 restriction factors such as APOBEC proteins22L1 activity was highly dynamic during reprogramming and hiPSC cultivation. Parental cells, early hiPSC passages, later hiPSC passages and re-differentiated cells presented grossly different levels of L1 expression. As corroborated by RC-seq, genotyping PCR and qPCR, the majority of retrotransposition in hiPSCs likely took place during or immediately after reprogramming, where we observed a peak in expression of the L1 mobilization machinery. As a result, each detected variant could affect substantial hiPSC subpopulations. Interestingly, major induction of L1 mRNA and protein expression, far in excess of that seen in hESCs and neural stem cells13de novo L1 insertions reported here were full-length was consistent with 2/3 of the engineered L1 de novo insertions characterized by Wissing et al. also being full-lengthde novo insertions in hiPSCs is unexpected as only ∼15% of L1 copies in the human reference genome and <1% of somatic L1 insertions identified in tumours are full length333650de novo L1 insertions found in hiPSCs. The mechanism of L1 5′ truncation is not fully understood. On one hand, the preponderance of 5′ truncated L1 copies in the genome has long been explained by an inability of the L1 reverse transcriptase encoded by L1 ORF2p to copy the entire template L1 RNA, either due to premature dissociation of the L1 reverse transcriptase from its RNA or competition from an unknown cellular RNase that digests the L1 RNA before completion of reverse transcriptionde novo L1 insertions in human neural stem cellsThat 4/7 of the de novo L1 insertion reported here integrated in a protein-coding gene expressed in pluripotent cells. In one case, we identified an L1 insertion (L1-dn13) that arose during hiPSC cultivation and integrated into an intron of the gene CADPS2. It remains to be determined whether acquisition of L1-dn13, and a concurrent reduction in CADPS2 expression, imbued carrier hiPSCs with a selective advantage in vitro. Furthermore, it remains unclear why transcription of the CADPS2 allele lacking L1-dn13 was reduced by >90%. To speculate, it is possible that CADPS2 expression involves a direct or indirect positive feedback loop where, for example, transcription from CADPS2 reinforces open chromatinCADPS2 expression caused by L1-dn13 could hence have a strongly negative effect on transcription from the wild-type CADPS2 allele.Each Alu and SVA insertions can alter cellular phenotype and are associated with numerous instances of human disease2345In closing, it is notable that intronic L1, OCT4, SOX2, KLF4 and c-myc) or OSKML (OSKM+LIN28) expression cassettes5 cells per well) were transfected by nucleofection (Lonza) according to the manufacturer’s instructions. In each transfection, 2 μg of transposon plasmid (pT2-OSKM or pT2-OSKML) and 0.2 μg of CMV- SB100X vector , 20% Knockout Serum Replacement (Gibco), 1 mM L-glutamine (Biochrom AG), 50 μM β-mercaptoethanol and 0.1 mM nonessential amino acids. The medium was replaced every day. Newly formed hiPSC colonies were picked, transferred to Matrigel-coated 24-well plates, and expanded for 4–6 days in MEF-conditioned ESC medium. Subsequently, cells were trypsin dissociated, plated onto feeder cells and cultivated in ESC medium.hiPSC lines hiPS-SB4 (hFF-T2-OSKM) and hiPS-SB5 (hFF-T2-OSKML)/hiPS-SB5.1 (hiPS-OSKML#6) were generated by reprogramming HFF-1 cells (ATCC-Number: SCRC-1041) using Sleeping Beauty (SB) transposon-based plasmids pT2-OSKM or pT2-OSKML which contain polycistronic OSKM  based on their positivity for SSEA4. Six SB-OSKML hiPSC clones and one SB-OSKM hiPSC clone were then characterized for pluripotency and differentiation potential as describedThe lines hiPS-CRL1502 ref. , hiPS-CRhttp://www.geneastemcells.com.au). It formed well-defined colonies with compact cells displaying a high nuclear to cytoplasmic ratio and prominent nucleoli. Karyotype analysis  did not uncover any abnormalities at passage 42. HESG cells express pluripotency markers Nanog, Oct-4, Tra1-60 and SSEA4, stain positive for alkaline phosphatase and form teratomas. As for hiPSCs, hESCs were grown on gelatin-coated six-well plates (Greiner) on inactivated mouse embryonic fibroblasts . MEFs were expanded and mitotically inactivated by γ-irradiation with a Cesium source with 30 Gy after 3–7 passages, and stored in liquid nitrogen until further use. After thawing, MEFs were seeded at a density of 6 × 105 cells per well of a six-well plate. hESC medium was replaced daily and cells were passaged at a 1:2 dilution every 5 days using splitting medium  in KO-DMEM).hESC lines H1, H9 and HES-3 were purchased from the WiCell Research Institute  and Cythera Inc. . The H1 line was used exclusively for the isolation of cell lysate that was loaded as positive control of the immunoblot analysis in hiPSCs were washed once in PBS containing 0.5% bovine serum albumin, and incubated for 30 min with allophycocyanin-conjugated anti-human SSEA4 antibody (R&D Systems). In all samples an anti-mouse Sca-1 (Ly-6A/E)  antibody was employed, for gating out the positively labelled mouse feeder cells. Samples were analysed and sorted using an Aria High Speed Cell Sorter (Becton-Dickinson).−1; Gibco) per well of a six-well tissue culture plate. Next, cells were resuspended in 750 μl of ESC medium, transferred to a 15 ml conical tube and centrifuged at 800 r.p.m. in a Heraeus Multifuge 4KR for 3 min at room temperature. Subsequently, medium was removed, cells were resuspended in 3 ml of ESC medium without FGF2 and cultured for 1–16 days in T25 flasks (Greiner) containing 10 ml of ESC medium without FGF2. At the indicated time, embryoid bodies were harvested and cytoplasmic RNA was isolated as described below. Passage 10 of the hiPSC line hiPS-SB5.1 was cultured in one well of a GeltrexTM-coated six-well culture dish, and treated with collagenase IV (1 mg ml−1) for 5 min. Cells were washed with warm PBS twice, and fed with 1 ml embryoid body formation medium ) and split into small cell clumps. hiPSC colonies were then dissociated with collagenase IV (1 mg ml−1) for 5 min, and split into small cell clumps. Cell clumps were transferred into three 10-cm low-attachment dishes and fed with embryoid body medium. The medium was changed every 2 days. Embryoid bodies were cultured for 8 days in total. Embryoid bodies were collected by sedimentation under gravity from three dishes on day 0 (undifferentiated hiPSCs), 2, 4, 6 and 8, respectively . For each time point and transcript to be quantified, qRT–PCR analyses were done in triplicate. qRT–PCR for pluripotency/differentiation markers was carried out using Power SYBR Green PCR Master Mix (Applied Biosystems) on the ABI7900HT sequence detector (Applied Biosystems), and data was normalized to GAPDH expression. qRT–PCR for L1 was performed with ABsolute QPCR Mix (ABgene), and data was normalized to 18S rRNA expression.6 to 3 × 107 somatic cells, hiPSCs or embryoid body cells using the RNeasy Midi Kit  according to the manufacturer’s instructions. Cytoplasmic RNA (0.5–1 μg) was incubated with 2 U of RNAse-free DNaseI  for 30 min at room temperature. DNAseI digestion was stopped by adding 2 μl of 25 mM EDTA and incubation for 10 min at 65 °C. DNAseI-digested cytoplasmic RNA (0.1–0.5 μg) was used for cDNA synthesis using the SuperScript III First-Strand Synthesis Kit (Invitrogen) in combination with a Random Hexamer Primer  according to the manufacturer’s instructions. Quantitative real-time PCR was carried out in ABgene plates using an Applied Biosystems 7900HT Fast Real-Time PCR System. The primer and probe combination L1 5′UTR#2  spanning exon sequences (Cytoplasmic RNA was extracted from 5 × 10R#2 ref.  was usedequences . Cycling+-Nylon membrane (Amersham) by overnight capillary transfer using 10 × SSC as transfer buffer. A total of 4 μl RiboRuler High Range RNA ladder  were loaded as size marker. After crosslinking the RNA onto the membrane by ‘baking’ at 80 °C for 2 h, the membrane was prehybridized overnight in 50% Formamide/4xSSC/1%SDS/2 × Denhardt’s at 42 °C. The full-length L1 mRNA-specific probe was generated by PCR amplification of a 1299-bp L1 fragment ranging from position numbers (pos.) 58–1356 of a full-length L1 element by using primers L1_FW1 and L1_RV1 (RPΔCMV32P]dCTP by applying the Nick Translation System (Invitrogen) according to the manufacturer’s instructions.Total RNA was isolated from the cell lines HFF-1, 2102Ep ref. , HES-3 ad L1_RV1  and pJM1V1 RPΔCMV and plasV1 RPΔCMV as templAfter denaturing the probe for 10 min in boiling water and subsequent incubation for 10 min in ice water, the probe was added to the hybridization buffer (50% Formamide/4 × SSC/1% SDS/1 × Denhardt’s) and the membrane was incubated in the probe-containing hybridization buffer overnight at 42 °C. Subsequently, the membrane was subjected to two 5 min low-stringency washes (2 × SSC) at room temperature and one 30 min high-stringency wash (2 × SSC/0.5%SDS) at 65 °C. The membrane was stripped by being boiled for 30 min in a solution of 10 mM tris-HCl (pH 7.5)/1 mM EDTA/1 mM SDS. The hybridized membrane was exposed to X-ray films for 5–10 days with intensifying screens.http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker. Next, the fraction of unmethylated CpG sites was calculated by comparison to a consensus L1-Ta sequence. In addition, each individual sequence was compared to L1.3 and only the sequences with the highest homology to this sequence were used to plot methylation data in single clones .Bisulfite DNA sequencing analyses were performed as previously described20e clones . The pro−1 collagenase type IV/DMEM and washed subsequently in 1 × PBS. Cells were spun down, resuspended in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% Glycerin, 1% Triton X-100; 2 mM EDTA, 2 mM EGTA, 40 mM β-Glycerolphosphate disodium salt hydrate, 50 mM NaF, 10 mM Na4P2O7, 200 μM Na3VO4, 2 mM DTT; 1 × complete protease inhibitor cocktail (Roche Applied Science)), homogenized by passing the lysate ten times through a 26 G needle, and lysates were cleared by centrifugation. A total of 50 μg of each protein lysate were boiled in 3 × SDS sample buffer (NEB), loaded on 4–12% Bis/Tris gels (Invitrogen), subjected to SDS–polyacrylamide gel electrophoresis, and electroblotted onto nitrocellulose membranes. After protein transfer, membranes were blocked for 2 h at room temperature in a 10% solution of non-fat milk powder in 1 × PBS-T ), washed in 1 × PBS-T, and incubated overnight with the respective primary antibody at 4 °C.hiPSC colonies were detached from their tissue culture dish by incubation with 250 μl of a 1 mg mlL1 ORF1p and Oct-4 proteins were detected using the polyclonal rabbit-anti-L1 ORF1p antibody #984 ref.  at a 1:2hiPSCs as well as their parental HFF-1 or hCBEC cells were grown on glass cover slips in 12-well plates. Cells were washed with 1 × PBS, fixed with 4% paraformaldehyde in 1 × PBS (pH 7.4) for 15 min at room temperature and permeabilized with 1% Triton X-100 (Sigma) in 1 × PBS for 10 min at room temperature. Subsequently, cells were washed thrice for 2 min in 1 × PBS. Cells were blocked by incubation with 5% (w/v) BSA/0,1% Triton X-100/1 × PBS (pH 7.4) for 30 min at room temperature followed by incubation with the respective primary antibodies, which are listed in 6 cells from each hESC and hiPSC line and their respective parental cells using DNAzol Genomic DNA Isolation Reagent  according to the manufacturer’s instructions. RC-seq and subsequent computational analyses were performed as described using the hg19 reference genome sequencede novo insertions supported by at least two unique amplicons separated by ≥5 nt (the minimum threshold for reporting) is provided in de novo L1 insertion with ≥2 unique reads at a given sampling depth, normalized to the corresponding false negative rate identified above and then determined the cumulative sum of this distribution for frequencies of 5–100%, leading to an estimate of ∼1 de novo L1 insertion per hiPSC. We did not consider de novo L1 insertions carried by fewer than 5% of hiPSCs in this estimate as none of the validated examples were routinely identified at that sampling depth. We also did not analyse the L1 mobilization rate in hESCs or the Alu or SVA rate in hiPSCs or hESCs due to the small number of confirmed true positive examples.Genomic DNA was isolated from 1 × 10de novo L1 insertions at the 5′ end of genes was performed by random sampling of genomic coordinates, with respect to RefSeq annotations. 1 × 106 permutations were performed and in 6,000 instances the average position was less than the 20th percentile of gene length, indicating P<0.006.A permutation test showing enrichment for validated de novo insertions  detected by RC-seq were first assayed with PCR using a standard empty site/filled site genotyping assay. Primers were positioned on either side of the insertion site so that the predicted PCR product of the empty site covered <300 bp. Additional retrotransposon specific primers were designed and paired with the existing insertion site-specific primers if required. In cases where an insertion was detected by RC-seq at one terminus only, PCR and capillary sequencing were applied to the remaining end to resolve integration site structure. PCR reactions contained 0.125 μl Crimson Taq (New England Biolabs), 5 × PCR-buffer, 10pMol of each Primer, 10 mM dNTPs and 10–20 ng genomic template DNA in a total volume of 25 μl. The following cycling conditions were used: 95 °C for 2 min, then 35 cycles of 95 °C for 30 s, 58 °C for 30 s, 68 °C for 40 s, followed by a single extension step at 68 °C for 5 min. Optimization in some cases required adjusted annealing temperatures and cycle number. PCR products of the correct size n repeat and Alu-like region, respectively. Combinatorial use of these genome-SVA primer pairs did either not result in a PCR product or generated non-specific products. For a complete list of used primers see de novo insertions  were confirmed by PCR as de novo. Six additional insertions were determined as germline insertions, already present in the parental cell line or an early hESC passage. Control genotyping PCR of the single-copy gene GAPDH in genomic DNA preparations of parental and hiPSC lines used for RC-seq and PCR validations of de novo insertions is presented in Seventeen ect size  that wer2 3′ end , at the de novo insertions L1-dn13 and L1-dn14 within the hiPS-SB4 culture, we applied real-time qPCR using TaqMan fluorogenic probes. To this end, genomic DNA was isolated using 1 ml DNAzol Genomic DNA Isolation Reagent  from 1 × 106 cells, according to the manufacturer’s instructions. A total of 100 ng of genomic DNA was used for quantitative real-time PCR (qPCR). Primer and probe combinations specific to the genomic 5′ junctions of the de novo insertions L1-dn13 and L1-dn14  was used. Cycling conditions were: 95 °C for 15 min (one cycle), 95 °C for 15 s and 60 °C for 1 min (40 cycles). For analysis of real-time and end point fluorescence, the software SDS version 2.3 as well as RQ manager 1.2 (Applied Biosystems) were used.To determine relative copy numbers of  L1-dn14  were usede novo retrotransposition event, were magnetically separated from feeder cells by applying a Feeder Removal Kit  according to the manufacturer’s instructions. hiPSCs were counted and seeded on feeder-coated 96-well plates  at a cell density of one cell per well or 0.3 cells per well. hiPSCs were grown for 24 h in the presence of 10 μM ROCK inhibitor . Subsequently, cells were cultivated until they formed a single colony per well. Single colonies were transferred to feeder-coated 12-well plates (Thermo Fisher/Nunc) and further expanded. To isolate genomic DNA from each clone, cells were harvested after collagenase IV treatment, centrifuged, washed and pelleted again. Genomic DNA was isolated as described in the previous paragraph. Genotyping PCR conditions applied to screen for the presence of the L1-dn13 insertion and to demonstrate its presence/absence  and primers located 50 bp upstream/downstream the insertion site (available upon request). For each PCR we used: 0.3 μl Expand Long Template Taq (Roche), 1 × buffer#1, 400 μM dNTPs, 1 μM each Primer and 300 ng genomic DNA in 50 μl per tube. Cycling conditions were: 95 °C for 5 min, then 30 cycles of 95 °C for 1 min, 56 °C for 30 s, 68 °C for 6 min, followed by a single extension step at 68 °C for 10 min. To avoid the generation of mutations that may lead to retrotransposition defective elements, we conducted at least four independent PCRs per L1. PCR products were resolved on 0.9% agarose gels, and fragments of the expected length of ∼6 kb representing potential full-length L1 elements were excised and purified using a Qiaquick kit (Qiagen) and cloned in the Topo-XL plasmid (Invitrogen). Each of the cloned PCR products carrying full-length L1 elements L1-dn4, L1-dn6 and L1-dn14 were sequenced  supplemented with L-glutamine, Penicillin/Streptomycin, and 10% fetal bovine serum  and passaged using Trypsin 0.05% (GIBCO-Invitrogen). 104 HeLa cells per well were plated in triplicate using six-well tissue culture plates. After 18 h, cells were transfected with 1 μg per well of plasmid using 3 μl of Fugene6 (Promega) following manufacturer’s instructions. Next day, medium was replaced and cells cultured for five additional days. Six days after transfection, Blasticidin-S (Invitrogen) was added to a final concentration of 10 μg ml−1 and cells were cultured for seven days in the presence of the antibiotic. Next, plates were fixed and stained with crystal violet, and foci counted manually.Retrotransposition assays in HeLa cells were conducted as described previously94652R2=0.79). Analyses were performed with SAS/STAT software, version 9.2 SAS system for Windows.The statistical evaluation of relative L1 mRNA levels determined by qRT–PCR was performed by ANOVA, using Bonferroni correction for multiple comparisons with the same control group. Reduction in full-length transcript levels in the embryoid body time kinetics experiment was evaluated by means of Linear Regression for data from day 0 to day 8 (Accession codes: The RC-seq FASTQ data have been deposited in the Sequence Read Archive (SRA) under accession code PRJEB3191.How to cite this article: Klawitter, S. et al. Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells. Nat. Commun. 7:10286 doi: 10.1038/ncomms10286 (2016).Supplementary Figures 1-15, Supplementary Tables 1-3, Supplementary Methods and Supplementary ReferencesEndogenous retrotransposon insertions in analyzed hiPSC and hESC lines identified by RC-seq


This study, and in particular the suggestion of a mechanism for epitaxial growth of oxides on graphene, offers new directions to exploit the development of oxide/graphene multilayer structures and devices.The transfer process of graphene onto the surface of oxide substrates is well known. However, for many devices, we require high quality oxide thin films on the surface of graphene. This step is not understood. It is not clear why the oxide should adopt the epitaxy of the underlying oxide layer when it is deposited on graphene where there is no lattice match. To date there has been no explanation or suggestion of mechanisms which clarify this step. Here we show a mechanism, supported by first principles simulation and structural characterisation results, for the growth of oxide thin films on graphene. We describe the growth of epitaxial SrTiO Therefore, it is necessary to develop a method for deposition of atomically uniform oxide layers without damaging the underlying graphene and without creating high interface defect density between the graphene and oxide materials3 (STO) thin films on graphene, which were prepared by chemical vapour deposition (CVD) and subsequently transferred onto STO and MgO substrates. The suggested mechanism is supported by density functional theory (DFT) modelling and verified by structural and electrical characterisations.Herein, we propose a growth mechanism that explains the formation of epitaxial SrTiO3 (Gr/STO) interface was modelled in a 2D-periodic square cell of side 8.85 Å containing 10 STO formula units representing four atomic layers of the (100) surface in a 2 terminations in a 2 surface layer. The average binding energy of the graphene to the oxide surfaces is 56 meV per C-atom. Calculation of the electrostatic field above the sheet establishes that the corrugation in the electrostatic potential is negligible suggesting that a complete graphene sheet will screen oxide overlayers from the direct influence of the substrate. This is confirmed by an explicit calculation of the corrugation potential computed by adding a periodic SrO sheet above the graphene layer and computing its energy on a 10 × 10 grid of points as it slides across the surface (perpendicular atomic coordinates optimised for each configuration). The resultant energy surface has a corrugation of less than 1 meV per carbon atom. Undisturbed through-graphene layer epitaxial growth mediated by wrinkling of the graphene sheet or electrostatic interactions is therefore highly unlikely.The graphene/SrTiOnterface . In the The ease with which the sheet slides across the oxide surface suggests an alternative mechanism for epitaxial growth. Defects in the sheet introduced during the graphene growth or transfer provide reaction centres for the adsorbing oxide and may allow bridge structures to form between the substrate and the oxide overlayer. In this scenario, an oriented single crystal STO film on a graphene layer might be supported by epitaxial pillars grown through the defects in the graphene layer (e.g. grain boundaries) . Within −8 Torr). The thin film growth was monitored using reflection high energy electron diffraction (RHEED). The specular RHEED intensity shows a sharp decrease and a recovery during deposition of the first unit cell, illustrated in In this study, 100 nm STO thin films were grown on Gr/MgO and Gr/STO by PLD at 850 °C in ultra-high vacuum (~10l) peaks from 2θ of 20° to 120° (not fully shown here). This indicates that STO was epitaxially grown on Gr/STO. To further prove the growth of STO on graphene, STO films were deposited on Gr/MgO. The (002) STO peak clearly appeared on the XRD pattern of STO/Gr/MgO, shown in D, G and 2D) as shown in D peak intensity (ID) at ~1350 cm−1 is related to the amount of disorder in graphene layers22−1, denoted by G, is caused by the doubly degenerate zone centre photon E2g mode242D peak located at around 2690 cm−1, is induced by a double resonance electron-phonon scattering processG peak and 2D Peak) on the Raman spectrum of Gr/MgO. The absence of D peak reveals a higher crystalline quality of the graphene layer2D peak intensities, I2D/IG, on Gr/STO and Gr/MgO are approximately 2, which are characteristic for monolayer graphene.Raman spectroscopy of random regions on the samples was undertaken to confirm the presence of graphene after STO growth. The Raman spectrum of Gr/STO has three prominent peaks  or even to less than 1 (STO/G/STO sample), see more details in 2 molecules27282931G and 2D peaks along with decrease of the full width at half maximum (FWHM) value of G peak27I2D/IG ratio is attributed to the compressive strain in the graphene and formation of multilayer graphene caused by the construction of STO pillar structures on the defects.Raman spectra of STO/Gr/STO and STO/Gr/MgO samples are not very different from those of samples without STO thin films. No appreciable peak position shift is detected. But the full width at half maximum (FWHM) values of film/Gr/STOsubstrate structure. The calculated geometry suggests that a distance about 0.3 nm is optimal. Also, there are well detected areas  thin films on graphene transferred onto STO and MgO substrates was revealed. The initial local defects in the graphene layer (e.g. grain boundaries) act like bridge-pillar spots that enable the epitaxial growth of STO thin film over the graphene layer. The growth of epitaxial STO pillar structures increases the interfacial sheer stress, which results in partial folding of the graphene layer. The results were confirmed by DFT modelling, HRTEM studies, XRD and Raman spectroscopy. The I-V measurement of the graphene after the STO film growth explicitly confirmed the conservation and the existence of the graphene layer after the deposition process. The evidence provided suggests that it is indeed possible to grow epitaxial oxide films on graphene.2 gas for 10–60 minutes. The growth was conducted in a high temperature tube furnace by introducing 40 sccm of CH4 gas at a total pressure of ~8 mbar for 10–30 minutes. Samples were unloaded from the quart tube after cooled to RT. Subsequently, a Poly(methyl methacrylate) (PMMA) layer was spin-coated on graphene to facilitate the transfer of graphene onto STO and MgO substrates. Then, the copper foils were etched. Finally, the PMMA/graphene layers were transferred onto STO and MgO substrates.Single layer of graphene was grown on copper foils at temperatures up to 1000 °C by CVD. Before growth, the copper foils (25 μm thickness) were pre-treated with acetic acid solution (50%) for 20 minutes and annealed at 1000 °C under 80 sccm H2 termination.However, before transferring graphene onto STO substrate, the surface of STO substrate was thoroughly cleaned in ultrasonic bath using acetone, isopropanol, and deionized water, then dipped in buffered HF etchant. The etched STO substrates were annealed at 1000 °C for 1 hour before transfer. The treated surface of the STO substrate is believed to be atomically flat and dominated by TiOAfter the graphene transfer, it is vital to eliminate the deleterious effects of the additional PMMA layer on top of the graphene layer. Therefore, as transferred samples were heated to 400 °C for 1 hour in ultrahigh vacuum (UHV) chamber to remove undesired PMMA and chemisorbed impurities, such as oxygen, carbon dioxide, carbon monoxide and water.−8 Torr) using a KrF excimer laser (λ = 248 nm). A stoichiometric single phase STO target was ablated with a laser fluency of 2 J/cm2 at 2 Hz. After deposition, as grown film was slowly cooled down to 600 °C at a rate of 10 °C/min in UHV and was subsequently in-situ post-annealed at 300 Torr O2 pressure for 1 hour to compensate the potential oxygen loss during the growth.Gr/STO and Gr/MgO samples were annealed at 850 °C in vacuum before deposition. To prevent destruction of the graphene layer via thermal oxidation, STO thin films (100 nm) were grown on Gr/STO and Gr/MgO at 850 °C by PLD in UHV .The surface of graphene was investigated with the aid of a high resolution field emission scanning electron microscope (FEG-SEM) LEO GEMINI 1525 FEG-SEM with an accelerating voltage of 5 kV and working distance of ~6.5 mm. Cross-sectional samples were prepared using an FEI Helios dual-beam and final thinning was done at 500 eV with an Ar ion beam on a Fischione Nanomill. Transmission electron micrographs were taken using an aberration-corrected (at the image plane) FEI Titan 80–300 scanning/transmission electron microscope (S/TEM). All analysis was done with electron energy of 80 keV.How to cite this article: Zou, B. et al. Growth of Epitaxial Oxide Thin Films on Graphene. Sci. Rep.6, 31511; doi: 10.1038/srep31511 (2016).


There are two critical challenges which determine the array density of 3D RRAM: 1) the scaling limit in both horizontal and vertical directions; 2) the integration of selector devices in 3D structure. In this work, we present a novel 3D RRAM structure using low-dimensional materials, including 2D graphene and 1D carbon nanotube (CNT), as the edge electrodes. A two-layer 3D RRAM with monolayer graphene as edge electrode is demonstrated. The electrical results reveal that the RRAM devices could switch normally with this very thin edge electrode at nanometer scale. Meanwhile, benefited from the asymmetric carrier transport induced by Schottky barrier at metal/CNT and oxide/CNT interfaces, a selector built-in 3D RRAM structure using CNT as edge electrode is successfully fabricated and characterized. Furthermore, the discussion of high array density potential is presented. As one of the most promising emerging non-volatile memory (NVM) devices, oxide-based resistive switching memory (RRAM) has attracted significant interests due to the super endurance, fast switching speed, low power consumption and good CMOS compatibility12345681011y and Leffective represent the thicknesses of the metal plane and the effective perimeter which is in touch with resistive switching layer at horizontal direction. For traditional metal plane electrode based 3D RRAM, since it has cylindrical shape with surrounding electrode, the Leffective could be calculated as:Where Lx,pillar, Lx,RRAM, Lx,S.L. represent thicknesses of pillar electrode, resistive switching layer and selector layer respectively. Previous studies on planar RRAM structure have already shown that 3 nm RRAM device could deliver normal resistive switching performances14y? 2) How to find an efficient method to integrate a selector device? Similar to 2D crossbar structure, 3D RRAM array also has sneak path current issue, as shown in 10x,S.L.) in those approaches are even larger than that of switching layer , which limits the size scaling in horizontal directionWhere LRecently, carbon based low dimensional materials, including carbon nanotube(CNT) and graphene, have drawn significant scientific and technological interests as the emerging interconnect solution18212O5-x/TaOy RRAM cells are fabricated using monolayer graphene as the edge electrode. 2 substrate by an electrochemical approach2O5-x/TaOy RRAM devices using monolayer graphene as the edge electrode. Two-layer 3D Ta2O5-x/TaOy RRAM with graphene edge electrode is shown in y oxide layer contacts with graphene layer during deposition process, the edge of graphene could be oxidized. The previous studies have shown that, in the graphene/Ta2O5-x/TaOy/graphene system, a certain concentration of epoxide groups were grafted onto the basal plane of grapheney and graphene. Serving as an internal resistor, this high resistance region helps control the overshoot current and achieves self-compliance property during SET and forming operations. 4 s at 85 °C.The electrical performance of two-layer 3D Ta2O5-x interface, other than graphene/TaOy interface. During forming process, the oxygen vacancies in TaOy layer move to Ta2O5-x layer, at the Pt/Ta2O5-x the conductive filaments are formed. On the graphene/TaOy side, there are a lot of oxygen vacancies in TaOy which has good conductivity. Electrons could transport from graphene to TaOy easily and enough current could be supplied. Since the graphene electrode has little effects on this process, it is possible that the filament size is still in several-nanometers order, which could be larger than graphene electrode.Furthermore, It is necessary to investigate the mechanism when the edge electrode scaling to sub-nanometer. Therefore, The temperature dependent transport characteristics in HRS and LRS are studied to understand the conduction mechanism. The above physical characterizations and electrical results prove the proposed 3D RRAM cell could switch successfully with monolayer graphene as edge electrode. This ultra-small feature size (~0.3 nm) confirms the 3D RRAM could scale down to sub-nanometer in vertical direction.2x103D RRAM has more challenges of scaling down in horizontal direction. Due to the sneak path current issue, as shown in 2O5-x/TaOy RRAM using CNT as the edge electrode. The effective area Seffective in this case is further reduced, which closes to the magnitude of CNT cross-section area. The fabrication process is similar to that of 3D RRAM with graphene edge electrode, as shown in 2 substrate directly by CVD method2 insulator layer using HF chemistry which wouldn’t damage the CNT, as shown in A novel approach of using CNT as the edge electrode and a self-integrated selector is proposed and devices are fabricated. 2O5-x/TaOy RRAM with CNT edge electrode. Both metallic and semiconducting CNTs were investigated. The metallic CNT with TaOy and Sc electrodes shows a near-ohmic behaviour as shown in 4 s at 85 °C, as shown in y. At reverse cut-off state with positive voltage on edge electrode and negative voltage on pillar electrode, the Schottky barriers block the hole transport at Sc/semiconducting CNT end and the electron transport at TaOy/semiconducting CNT end, as shown in the inset of y could serve as the built-in bi-directional selector for 3D RRAM without inserting any additional selector layer.For semiconducting CNT as edge electrode case, a totally different transport property was observed, as shown in 4 s at 85 °C at 10 GΩ and 100 MΩ with 5 V read voltage, as shown in By choosing the metal contact Sc and TMO deposition sequence, the built-in selector could reduce the over-shoot current and sneak-path current simultaneously: During SET process with negative voltage on pillar electrode, the build-in selector is under reverse bias, which reduces the over-shoot current and achieve better self-compliance property. While in RESET process, the selector under forward bias supplies enough driving current to assist the resistive switching process. Since the 3D RRAM has been experimentally demonstrated at single cell level, it is important to evaluate the performance potential at the array level. Next, a simulation study is conducted to evaluate the 3D RRAM architecture with metal plane/2D/1D nano-materials. A resistor network model is constructed using SPICE method assisted by MATLAB. Three types of 3D RRAM cells, using Pt edge electrode, graphene edge electrode and semiconducting CNT edge electrode, have been compared based on measured data. Moreover, the interconnect resistance between neighboring cells is taken into account, following previous literature4 bits for device with Pt edge electrode. In contrast, for graphene and CNT edge electrode devices, the voltage degrade to 2 V when the array sizes increase to 108 bits and 1010 bits. The decrease of write access voltage mainly comes from the interconnect resistance. With the array size increase, the equivalent resistance of interconnect is comparable with RRAM cells, which causes the divided voltage on selected cell decrease. Therefore the 3D RRAM with Pt edge electrode has the limited array size due to small HRS/LRS resistances. For 3D RRAM cells with graphene and CNT edge electrodes, the HRS/LRS resistances are 1000X larger. This property offers much large array size without write access voltage degradation.m = 80 mV is set as the criterion6 bits which is still not good enough for high density applications. With semiconducting CNT edge electrode case, owing to the built-in selector benefit, the sneak path currents could be reduced efficiently and less voltage drops on other unselected cells. As a result, more than 108 bits array size could be achieved.The simulation results show graphene/CNT edge electrode based 3D RRAM have better performance at array level, comparing with metal edge electrode based 3D RRAM. In addition, some non-ideal effects, such as non-uniformity, device imperfection, RTN noise, etc. are not included in the simulation. Although these effects will not affect the comparison results, they still need to be investigated in the future. The excellent vertical and horizontal scaling limits give 3D RRAM technology great potential in high density memory applications.2O5-x/TaOy RRAM cells with monolayer graphene as edge electrode are fabricated. 3D RRAM cells could switch normally with sub-nanometer electrode thickness. In horizontal direction, selector-layer free is realized by using 3D Ta2O5-x/TaOy RRAM with semiconducting CNT edge electrode. In such case, the Schottky barriers formed at CNT/Sc and CNT/TaOy contacts are served as the built-in selector. Based on the experimental and simulation results, different edge electrode material options are evaluated for high density application potential.In this work, 3D RRAMs with graphene or CNT edge electrodes are demonstrated, to explore the vertical and horizontal scalability. In vertical direction, two-layer 3D Ta2O5-x/TaOy RRAM with graphene edge electrode: 1) Synthesize and transfer the first layer graphene to SiO2 substrate. 2) Deposit 100 nm SiO2 insulating layer by RF sputtering. 3) Synthesize and transfer the second layer graphene to SiO2 substrate. 4) Deposit second SiO2 insulating layer. 5) Pattern by lithography and ICP dry etch with C4F8/Ar to form the drilled holes. 6) Deposit the Ta2O5-x/TaOy TMO layer by RF reactive sputtering. 7) Pattern by lithography and deposit 30 nm Pt as the pillar electrode. 8) Pattern by lithography and deposit the 40 nm Pd as the contact metal on graphene.3D Ta2O5-x/TaOy RRAM with CNT edge electrode: 1) Synthesize CNT on SiO2 substrate using CVD method. 2) Deposit 100 nm SiO2 insulating layer by sputtering. 3) Pattern by lithography and wet etch the drilled hole without damaging the CNT. 4) Etch the CNT using oxygen plasma in the drilled hole. 5) Deposit the Ta2O5-x/TaOy TMO layer by reactive sputtering. 6) Pattern by lithography and deposit 30 nm Pt as the pillar electrode. 7) Pattern by lithography and deposit the 40 nm Sc as the contact metal on CNT.3D Ta2 ambience. Then the reaction gases composed by CH4 and H2 were introduced into the growth quartz chamber with a total gas flow rate of 800sccm. The ratio of CH4:H2 is 0.0078:1 and the substrates were soaked in the reaction gas mixture for 60 minutes before cooling down step. The synthesized graphene films were transferred to SiO2 substrates by an electrochemical methodSynthesize and transfer the graphene edge electrode. Pt foils were heated to 1050 °C in H2 substrates. After the growth of CNTs, the samples were annealed at 80 °C in a low pressure environment for 5 min to make the substrates more hydrophobic.Synthesize the CNT edge electrode: The CNTs were synthesized by a CVD methodThe quality of graphene was characterized by Raman spectra (RENISHAW RM2000). The CNTs were observed using TEM (FEI Tecnai TF20) and SEM (QUANTA FEG450). The 3D RRAM cross-section structures were investigated by TEM, EELS (FEI Tecnai G2 F20) and FIB (FEI Nova600). The electrical characteristics were tested by Agilent B1500A semiconductor parameter analyzer and Agilent 81110A pulse generator with Cascade Summit 11000 probe station.How to cite this article: Bai, Y. et al. Stacked 3D RRAM Array with Graphene/CNT as Edge Electrodes. Sci. Rep.5, 13785; doi: 10.1038/srep13785 (2015).


We design a dual-band absorber formed by combining two cross-shaped metallic resonators of different sizes within a super-unit-cell arranged in mirror symmetry. Simulations indicate that absorption efficiencies greater than 99% can be achieved at two different frequencies under normal incidence. We employ a design scheme with graphene integration, which allows independent tuning of individual absorption frequencies by electrostatically changing the Fermi energy of the graphene layer. High absorbance is maintained over a wide incident angle range up to 50 degrees for both TE and TM polarizations. It thus enables a promising way to design electrically tunable absorbers, which may contribute toward the realization of frequency selective detectors for sensing applications. Metamaterial absorbers356791011141516171819202123252931323436363839404142In this paper, we numerically demonstrate, via highly confident numerical simulations, a dual-band metamaterial absorber where the two absorption bands can be independently tuned. This can be accomplished through the application of a voltage bias to modify the conductivity of the graphene layer that has been integrated into the metamaterial absorber super-unit-cell. The metamaterial device offers high absorbance over a wide range of incidence angles, and the independently tunable absorption bands might find applications in fields like tunable sensors and selective thermal emitters.ts = 0.285 μm, and the thickness of gold is tm = 0.1 μm. The periods along the x and y directions are Px = 14.4 μm and Py = 3.6 μm, the lengths of the bigger and smaller gold crosses are S1 = 3.0 μm and S2 = 2.0 μm, and their widths are W1 = 0.72 μm and W2 = 0.6 μm, respectively. The permittivity of the PTFE spacer is 2. The graphene layer is structured into interdigitated fingers with width Wg = 3.4 μm and spacing d = 0.2 μm. Two gate voltages, Vg1 and Vg2, are applied to the two graphene interdigitated finger sets in order to tune their Fermi energies independently.The schematic design of the proposed tunable metamaterial absorber is illustrated in S21 for transmission and S11 for reflection) and the absorbance was derived using The simulations were carried out using the well-established three-dimensional full wave electromagnetic solver, CST Microwave Studio 2012. We obtained the S parameters (ωp) and the scattering rate (Γ) for various Fermi energy as explained in Methods. By optimizing the thickness of the spacer ts = 0.285 μm, we obtained two absorption bands with peaks absorbance greater than 99%. When Ef = 0.2 eV, the two absorption peaks are positioned at wavelengths of 5.88 μm and 8.46 μm. Varying Ef from 0.2 eV to 0.8 eV, which could be accomplished by applying a voltage bias between the ground plane and graphene layer, both absorption peaks show blue-shift while the high absorbance is remained. The longer wavelength absorption peak shifts by ΔλS1 = 0.46 μm (5.4% shift), and the shorter wavelength absorption peak shifts by ΔλS2 = 0.15 μm (2.6% shift). The difference in the absorption wavelength shift is mostly due to the wavelength dependent change of the imaginary conductivity We first investigated a dual-band metamaterial absorber shown in H1 and H2, which apply to the corresponding two resonant structures. The wave reflected from the metamaterial absorber is quantified byIn order to further understand the blue-shift, we describe the metamaterial absorber as a combination of two independent transfer functions, E0 is the normally incident electric field. The absorbance is then given by ZG across the capacitor C. The entire circuit is backed by a transmission line of impedance ZL, representing the gold ground plane. The length of transmission line ZD is equal to the dielectric spacer thickness employed in the simulations and has the same effect of creating a resonant cavity between ZL and the RLC circuitwhere Zi to the wave incident from a transmission line of impedance Z0 = 377 Ω. The transfer functions are equivalent to field reflection coefficientsThe circuit presents a complex, frequency-dependent impedance ZL = 2 Ω was calculated using tabulated bulk gold conductivity valuesd~100 nm). The graphene impedance consists of real and imaginary parts,The load impedance Lk = 1/α is the kinetic inductance of the graphene and, being variable with  Ef, is the tuning mechanism of the absorber. By the equation for ZG it should be hypothesized that because ZG should play a minor role. However, this should not hold true at longer wavelengths and GR should begin to have a noticeable effect.The term Ri, Li, and Ci were determined empirically by matching the resonance frequency, Q-factor, and peak absorbance qualities of both resonances against simulations not involving any graphene layer. It was found that these factors were highly constrained if all three qualities were to be simultaneously matched. Numerical values for ZGi were calculated asThe values for Bi is a constant fitting parameter that is easily obtained by matching the magnitude of the frequency shift of the absorption peak over Ef = [0.2 : 0.8] eV. After ZGi was determined, the factors Ri, Li, and Ci were fine-tuned to give the final model parameters. These are summarized in where ZG shunted the entire RLC circuit or the LC sub-circuit, were also studied. In these cases, the absorbance bandwidth and/or the peak absorbance could not be kept constant in tuning over the entire Fermi energy range. By restricting the graphene effect to the capacitor alone, the net inductive reactance of Zi is largely maintained during tuning, being dominated by Li. Though the effective capacitive reactance changes, it has very little effect on the Q-factor. Thus, absorption bandwidth is almost completely unaffected during tuning. The net resistive impedance of Zi is also maintained, being dominated almost entirely by Ri for all values of Ef. This prevents any change in the peak absorbance during tuning. The third implication is the validation of the hypothesis that RG plays a required role in the tuning behavior at long wavelengths. By artificially zeroing RG in the circuit model, a negligible effect was observed on the CR2 resonant absorbance, whereas the CR1 resonant absorbance peak continually decayed during tuning.The equivalent circuit model results are summarized as the circles in Vg1 and Vg2, respectively. First, we calculated the absorbance spectrum of our absorber by changing the Fermi energy of graphene under the small resonators (CR2) via tuning bias voltage Vg2, while keeping the Fermi energy of graphene under the bigger resonators (CR1) fixed to be 0.5 eV. The simulated absorbance spectra are shown as the solid curves in λ2 = 5.88 μm to λ2 = 5.73 μm, a wavelength shift of 2.6%. An alternative expression of modulation is obtained by fixing the observation wavelength to λ2 = 5.88 μm. In this case one would observe an absorbance change λ1 = 8.23 μm arising from the bigger resonators remains nearly invariant. This implies that the two resonances are spectrally separated enough to prevent any noticeable mutual coupling. Thus we can independently tune the resonant frequency of the CR2 resonator by changing the Fermi energy of graphene under the small crosses. The same results can be obtained from the circuit model by fixing the Fermi energy of H1 to be 0.5 eV, which are shown as the circles in To investigate the independent tuning of each resonance, we adopt the device design shown in λ1 blue-shifts from 8.45 μm to 8.0 μm, a wavelength shift of 5.3%. An alternative expression of modulation is obtained by fixing the observation wavelength to λ1 = 8.45 μm. In this case one would observe an absorbance change Next, we calculated the absorption while varying the Fermi energy of the graphene layer under the bigger resonators (CR1) while fixing the Fermi energy of graphene under the smaller resonators (CR2) to 0.5 eV. The simulated absorbance spectra for various Fermi energies are shown as the solid curves in We have conducted additional simulations to investigate the behavioral dependence on angle of incidence. For these simulations we fix the Fermi energy of graphene under both resonators to be 0.4 eV, while maintaining all other aforementioned parameters. Absorbance as a function of wavelength and incident angle is shown in It should be noted that polarization-insensitive absorbers are highly desirable47484950We also note that the scattering rate of graphene may vary due to the impurities, defects, and interfacesIn conclusion, we showed that a dual-band absorber can be formed by using horizontally cascaded cross-shaped resonant structures at mid-infrared frequencies. Absorption peaks of greater than 99% were obtained under normal incidence, and high absorbance remained at large incident angles up to 50 degrees for both of the bands under TE and TM polarizations. Moreover, our metamaterial absorber structure enabled independent tunability of the two absorption bands by electrically controlling the Fermi energy of corresponding graphene layers. With its excellent performance, our design should be applicable in many fields such as tunable sensors and selective thermal emitters.tg = 0.5nm for all of our simulations. The effective dielectric constant of graphene can be calculated by tg is the thickness of graphene.The simulations were carried out using commercial Finite Element full wave electromagnetic solver CST Microwave Studio 2012. We employed a time-domain solver to obtain frequency-dependent absorption spectra in The complex surface conductivity of graphene can be calculated from the well-known Kubo formula and is described with interband and intraband contributions as32whereω is the angular frequency, Ef is the Fermi energy of graphene, Γ is the collision angular frequency, T is the temperature, kB is the Boltzmann constant, e is the elementary charge, and Ef, the intraband contribution dominates the graphene conductivity as the interband transitions are negligible due to Pauli blockingEf >0.2 eV for wavelengths of interest ranging from 5.5 μm to 10 μm. Then, graphene can be described by taking only the intraband part of the Drude-like conductivity as described in Eq. tg = 0.5 nm is the thickness of graphene layer. In our simulations, we set T = 300 K and scattering where The relationship of the Fermi energy 6 m/s), 2 and Kapton which require lower voltage bias while offering higher dielectric breakdown for our experimental demonstration. A recent demonstration shows that the gate voltage bias can be significantly reduced by applying a layer of ionic gel over graphene film with top gating architecturewhere How to cite this article: Zhang, Y. et al. Independently tunable dual-band perfect absorber based on graphene at mid-infrared frequencies. Sci. Rep.5, 18463; doi: 10.1038/srep18463 (2015).


The growth of graphene using resistive‐heating cold‐wall chemical vapor deposition (CVD) is demonstrated. This technique is 100 times faster and 99% lower cost than standard CVD. A study of Raman spectroscopy, atomic force microscopy, scanning electron microscopy, and electrical magneto‐transport measurements shows that cold‐wall CVD graphene is of comparable quality to natural graphene. Finally, the first transparent flexible graphene capacitive touch‐sensor is demonstrated. Therefore, a way forward to increase the throughput and reduce the production cost is to grow graphene in a cold wall CVD system which heats selectively only the Cu foils. Few types of cold wall CVD have been investigated so far for the growth of grapheneChemical vapor deposition (CVD) of monolayer graphene on copper2 fabricated on standard SiO2 substrates we measure a charge carrier mobility of 3300 cm2 V−1 s−1 and the quantum Hall Effect typical of single layer graphene. In contrast, the quality of graphene grown by hot‐wall CVD is often gauged only by carrier mobility,Here we report a completely new mechanism for the growth of graphene by resistively heated stage cold‐wall CVD which is markedly different from the growth mechanism of graphene in a hot‐wall CVD. Through a combined study of Raman spectroscopy, atomic force microscopy (AFM), and scanning electron microscopy (SEM) we elucidate the early stage formation of graphene by monitoring the transition from disordered carbon adsorbed on Cu to graphene. We also demonstrate for the first time 1) high‐throughput production, 2) ultralow cost, and (3) high quality monolayer graphene grown on Cu foils by resistively heated stage cold‐wall CVD. Our technique merges short deposition time (approximately few minutes) with high‐efficiency heating of a cold‐wall CVD system, resulting in ≈99% reduction in graphene production cost. The Raman spectra of our graphene films shows a low defect related peak and in devices with an area of 5600 μm high‐thr ultralowStudies of the growth mechanism of graphene on copper (using methane) in a hot‐wall CVD have thus far suggested the direct growth of 2D films involving several steps. The first step is the direct formation of 2D nuclei of grapheneFigure2/Si substrates using a wet transfer method.Our experiments show that the growth mechanism of graphene in cold‐wall CVD is markedly different from that of the hot‐wall CVD described above. Specifically, over a range of growth temperatures that we have investigated, we always observed a thick carbon film 100 nm), which forms in the early stages of the growth  ranging from 1 to 6 min. For all the samples we observe the characteristic peaks of sp2 bonded carbon atoms: the D‐peak at ≈1340 cm−1, the G‐peak around 1600 cm−1, the D′‐peak around 1620 cm−1, and the 2D‐peak at ≈2700 cm−1. For short tG  the D‐ and G‐peaks have considerable higher intensities than the 2D‐peak, which is typical of disordered carbon films.tG increases we observe changes in intensities, sharpness, and positions of the D and G peaks, and for tG > 4 min a well‐defined 2D‐band emerges. At the same time, AFM measurements show a reduction in the film thickness from 116 to 2.7 nm with increasing tG from 1 to 6 min , the full width at half maximum ) and the peak positions ). According to the three stage model for classification of disorder,ID/IG, FWHM and Pos(G) allow us to assess the ordering/amorphization in carbon materials ranging from graphite and amorphous carbontG = 1 min, the presence of a 2D peak with Pos(2D) = 2683 cm−1 and FWHM(2D) = 88 cm−1, the absence of a doublet in the D and 2D peaks, together with the overlap of G and D′ peaks indicate the formation of nanocrystalline graphite with no 3D ordering. Figure I2D and IG increase with increasing tG, whereas the ratio ID/IG decreases from ≈3.9 to 0.2 , and FWHM with increasing tG is consistent with the stage 1 ordering trajectory leading from nanocrystalline graphite to graphite. In this regime the size of sp2 clusters (La) increases with increasing ordering and can be estimated using the Tuinstra–Koenig relation ID/IG = C(λ)/La where C(532 nm) ≈ 4.96 nm.a ≈ 2 nm for tG = 1 min, which increases to La ≈ 25 nm for tG = 6 min as shown in Figure Lorentzian fitting of the D‐, G‐, and 2D‐peaks allows us to ascertain the structural ordering within the films by analyzing the band intensities  = 2678 cm−1 and FWHM(2D) = 30 cm−1 indicating the formation of monolayer graphene.For Figure2. Raman measurements confirm that these islands are composed of graphene. SEM analysis of films grown for 1000 and 1035 °C reveals a similar behavior of the saturation in island separation and a maxima in island size (see the Supporting Information). We observe that an increase in growth temperature leads to a reduction in the time required to achieve the maximum island size and to form a monolayer graphene as shown in the inset of Figure Ea = (Edes + Eatt – Ed – Ead)/2 and with the density of graphene islands Ni ≈ PCH4· exp(2Ea/KT), with Edes the desorption energy of a carbon monomer on the Cu surface, Eatt the barrier of attachment for the capture of a monomer by supercritical nucleus, Ed the activation energy of surface diffusion of a monomer, Ead the activation energy for dissociative adsorption of CH4 on Cu, PCH4 the methane partial pressure, K the Boltzmann constant, and T the growth temperature. Figure i has a dependence on growth temperature which is typical of the desorption controlled regime with an activation energy of 1.66 eV. The desorption model is also consistent with the formation of holes inside the islands at 8 min of growth , and FWHM for a 1 × 1 cm graphene film. Raman mapping measurements shown in the Supporting Information demonstrate the uniformity and high‐quality of the continuous films. The total processing time of this procedure is about 20 min (see the Supporting Information); this includes (1) heating up time for the CVD system from room temperature to the growth temperature, (2) Cu foil annealing time, (3) graphene nucleation and growth time, (4) cooling down time for the system to room temperature. The demonstrated processing time is significantly shorter than the processing time needed by hot‐wall CVD .−2 (see the Supporting Information). Compared with other CVD studies and neglecting the base cost of copper we see a reduction in the production costs of 98.83%–99.89%. This extraordinary reduction in the production cost, together with the possibility of reconstitution of high purity copper from etchant solutions by electrolysis that can yield up to 99% of the original foil,Having established the initial stages of graphene formation, we investigate the transition from graphene islands to a continuous film. Figure th time,  cooling 2/Si substrates. Using the parallel plate capacitor model we estimate the field effect mobility to be 3300 cm2 V−1 s−1 at 1.4 K and 2773 cm2 V−1 s−1 at room temperature. This mobility, measured across a large area device (≈0.05 mm2), is comparable to the mobility measured in smaller area devices of graphene either grown by CVD (50 μm222) and deposited on oxidized silicon substrates.2 V−1 s−1) multiplied by the area of the device (μm2). As shown in the Supporting Information, graphene grown by resistive heating cold‐wall CVD has Q ranging from 4 × 106 to 7.2 × 106, whereas most reports of monolayer graphene grown by hot‐wall CVD have Q ranging from 103 to 7 × 106. Hence cold‐wall CVD grown graphene has a narrow spread of Q stemming from a reproducible high quality growth process. This is in stark contrast to the spread of Q over three orders of magnitude reported for hot‐wall CVD grown graphene.To ascertain the quality of the electronic properties of graphene produced by cold‐wall CVD we characterized the charge carrier mobility in transistor devices fabricated on SiOvice ≈0.0 mm2, is Figure2, see inset) fabricated on standard SiO2/p‐Si substrates. The Si substrates are heavily doped and act as the gate electrode. By applying a voltage to the gate (Vg) we tune the carrier concentration from 3 × 1011 to 6 × 1012 cm−2. The charge neutrality point is at 0 V, indicating low residual doping levels in our samples. Figure σxy) against the applied Vg, taken at 13 T and at a temperature of 250 mK. Clearly developed conductance plateaus are visible when the Fermi energy is within a Landau level (N) that correspond to the conductance relationship σxy = (N + 1/2)4e2/h, typical of the half‐integer quantum Hall effect (QHE) of monolayer graphene with two‐fold spin and valley degeneracy.ρxx = 0 Ω where the Fermi energy is within the N = 0 Landau level for both electrons and holes, indicating that the graphene quality is high enough to observe the localization in the QH regime. At the same time, σxx shows well‐defined Shubnikov–de Haas oscillations which are periodic with the applied gate voltage. A color map of the differential Hall conductance plotted against perpendicular applied field and charge carrier concentration shows that Landau levels up to N = 6 are visible at high fields , therefore allowing us to demonstrate for the first time a flexible and transparent capacitive touch sensor using graphene for both the top and bottom electrodes.ρ ≈ 1.3 kΩ and the contact resistance <68 Ω. A detailed description of the fabrication procedure and electrical characterization is provided in the Supporting Information. The individual elements of the touch sensors are formed at the intersection between the graphene strips and each element represents a parallel plate capacitor. As pressure is applied to an element of the array, the dielectric elastically deforms reducing the spacing between the graphene electrodes resulting in an increase in capacitance. Figure Figure C = 6 pF was observed with a return to the original state after unloading. The sharp change in capacitance demonstrated indicates a fast responsivity to loading and unloading of the element. Finally we tested the flexibility and durability of the devices by bending the substrate and systematically measuring the resistance of the graphene strips across the device. Figure To test the responsivity of the device we periodically loaded and unloaded an element with a human finger and measured the change in capacitance shown in Figure In summary, we have shown a new growth mechanism of graphene by cold‐wall CVD, which starts with the formation of a thick carbon film in the early stages of the growth, that becomes progressively thinner with increasing the growth time and finally evolves into graphene islands. At the same time we demonstrate an extremely high‐throughput and cost efficient growth procedure for preparing high quality monolayer graphene using cold‐wall CVD. Finally, we use graphene as electrode material and demonstrate the first flexible and transparent graphene capacitive touch sensor using processing techniques that are compatible with existing transparent and flexible electronic technologies. Besides its importance for the quick industrial exploitation of graphene since cold‐wall CVD systems are found in semiconductor industries manufacturing plants, our work could lead to new generations of flexible electronics and offers exciting new opportunities for the realization of graphene‐based disruptive technologies.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.