the nonenzymatic replication 
 and transmission of genetic information 
 may have played an important role in the transition from prebiotic 
 chemistry to cellular life by enabling the evolution of selectively 
 advantageous ribozyme catalysts prior to the emergence of ribozyme 
 rna polymerases . the chemistry of nonenzymatic 
 oligonucleotide replication was extensively explored by orgel and 
 his students and colleagues . their most successful 
 model system involved the use of 2-methylimidazole - activated mononucleotides 
 to nonenzymatically copy short c - rich oligonucleotide templates . considerable progress has since been achieved 
 in demonstrating template - directed primer extension in the presence 
 of activated nucleotide derivatives . however , several challenges still need to 
 be addressed to show that nonenzymatic rna replication could be sufficiently 
 fast and accurate to allow for the evolution of new rna - encoded functions 
 within replicating protocells . foremost among these problems is the 
 slow rate of nonenzymatic primer extension on templates that contain 
 a and u residues . the rate of primer extension varies by more than 
 2 orders of magnitude depending on the specific nucleotide being added 
 to the growing chain . 2-methylimidazole - activated amp or ump ( 2-meimpa 
 or 2-meimpu ) are added to the 3-end 
 of a primer ( annealed to a complementary template ) at a much slower 
 rate than 2-meimpg or 2-meimpc . the rate of primer extension 
 with 2-meimpu is so slow as to be comparable to the rate of 2-meimpu 
 hydrolysis . a second major problem is 
 that g : u and a : c wobble pairing lead to a very high error rate in 
 nonenzymatic template copying , potentially 
 precluding the emergence of functional rnas such as ribozymes from 
 diverse sequence populations . we sought to address these challenges 
 by replacing u with su , a nucleobase known to significantly 
 stabilize base pairing 
 with a while modestly destabilizing wobble pairing with g. remarkably , the thione - mediated stabilization of the su : a base pair within an rna duplex is achieved without detectable 
 structural perturbation , suggesting that 
 the effect may be due at least in part to preorganization of the su - containing single strand . in 
 addition the larger and more diffuse electron cloud of sulfur vs oxygen 
 makes it more polarizable and hence may contribute to better stacking . replacing thymine with 2-thiothymine in dna is also stabilizing , and in rna we have therefore also explored the use of 2-thiothymine ribonucleotides 
 ( st ) in rna copying reactions . in addition to the 
 simple thermodynamic considerations , several 
 independent arguments support the possibility that su 
 or st might lead to improved nonenzymatic rna copying . nonenzymatic polymerization is most effective in the context of an 
 a - form helix . su substitution 
 in the anticodon loop of trna has been shown to increase the 3-endo 
 ( n ) conformer abundance of neighboring nucleotide sugars , and st stabilizes trnas from extreme 
 thermophiles , most likely through stabilization 
 of the 3-endo sugar conformation . we have previously shown 
 that monomers that are in the 2-endo conformation in solution 
 switch to the 3-endo state upon binding to an rna template . since the su mononucleotide has 
 been reported to exist in solution predominantly in the 3-endo 
 conformation , this preference should favor 
 binding to the template . finally , we have recently reported that nonenzymatic 
 primer extension in another system , in which primer , template , and 
 monomers are 3-amino-2,3-dideoxynucleotides , 
 exhibits enhanced rates and fidelity upon replacement of t with st . we report the effects of su and st substitutions on both the rate and fidelity of nonenzymatic 
 primer extension reactions in an all rna system . we show that these 
 substitutions in the activated monomer , but not in the template , contribute 
 to an enhanced ability to copy mixed sequence templates and that the 
 fidelity of copying is greatly increased . to examine 
 the effect of 2-thio substitution on nonenzymatic rna 
 primer extension , we measured rates of primer extension using both 
 2-thio substituted u and ribo - t monomers and templates . we used two 
 thiolated - nucleobase activated monomers , 2-thiouridine-5-phosphor-(2-methyl)imidazolide 
 ( 2-meimpsu ) and 2-thio-5-methyluridine-5-phosphor-(2-methyl)imidazolide 
 ( 2-meimpst ) , in nonenzymatic template - directed primer 
 extension reactions with fluorophore - tagged rna primers ( figure 1 ) . we used two types of templates for this study , 
 one type with six - nucleotide homopolymeric template regions u6 and a6 , and a second type with a single u or a 
 followed by five c residues , uc5 and ac5 ( figure 2 ; see also supporting information 
 section 1 for a complete list of oligonucleotides used in this 
 study ) . for pseudo - first - order rate determinations , primer extension 
 reactions were studied for a maximum of 50 min , much less than the 
 25 day half - time of hydrolysis of the activated nucleotides . pseudo - first - order rates and monomer  template dissociation 
 constants were determined from plots of the fraction of unreacted 
 primer as a function of time , for a series of activated nucleotide 
 concentrations ( table 1 ; figure 2 ; see also supporting information figure 
 1 ) . 2-meimpx analogues form watson  crick base pairs on 
 a complementary template and participate in template - directed primer 
 extension . ( b ) structure of thiolated uracil and thymine nucleobases 
 in 2-methylimidazole - activated nucleotides . we first considered the homopolymeric templates a6 , 
 u6 , su6 , and st6 . while primer extension with 2-meimpu on the a6 template was essentially undetectable at monomer concentrations 
 up to 175 mm ( kmax < 0.01 h ) , 2-meimpsu and 2-meimpst resulted in maximal 
 rates of primer extension ( kmax ) of 0.56 
  0.069 and 2.3  0.71 h , respectively 
 ( table1 ; figure 2a ; 
 see also supporting information figure 1b(1 ) ) . it is notable that the single - atom oxygen to sulfur substitution 
 resulted in readily observable primer extension ; furthermore , methylation 
 at the 5-position of 2-thiouracil increased the rate of primer extension 
 an additional 4-fold . in contrast , primer extension with 2-meimpa 
 on u6 , su6 , and st6 templates approached the lower bounds of experimental measurement 
 by this assay ( kmax = 0.027  0.0036 , 
 0.067  0.0081 , and 0.071  0.014 h , 
 respectively ) ( table 1 ; figure 2b ; see also supporting information figure 
 1b(1 ) ) . these low rates and the modest effects of 2-thiolation 
 and 5-methylation may reflect the poor stacking of u and modified 
 u monomers and thus poor preorganization of templates consisting of 
 multiple u ( or su or st ) residues in a row 
 into an a - type helical conformation . activated monomer and template 
 nucleotides are indicated at left . 200 mm hepes ph 7.0 , 0.5 m 
 p1 , 1.5 m template , on ice and ( 1 ) 1.0 m nacl , 200 mm mgcl2 ; ( 2 ) 1.0 m nacl , 200 mm mgcl2 , 40 mm 2-meimpg ; 
 ( 3 ) 100 mm mgcl2 , 40 mm 2-meimpg . kinetic studies of primer extension reactions . pseudo - first - order 
 rates were determined from the extent of primer disappearance as a 
 function of time , and the resultant observed rates were determined 
 as a function of activated nucleotide concentration to give kmax . reaction conditions : 200 mm hepes ph 7.0 , 
 0.5 m primer p1 , 1.5 m template , on ice . buffer 1 ( blue ) : 
 1.0 m nacl , 200 mm mgcl2 . ( a ) primer extension reaction on template t1 ( a6 ) with 2-meimpu * ( u * = u , su , or st ) . ( b ) 
 primer extension reaction on templates t2 ( u6 ) , t3 ( su6 ) , or t4 ( st6 ) with 2-meimpa . ( c ) primer extension reaction on template t8 ( ac5 ) with 
 2-meimpu * ( u * = u , su , and st ) and 40 mm 2-meimpg . ( d ) primer extension reaction on template t5 ( uc5 ) , t6 
 ( suc5 ) , and t7 ( stc5 ) with 2-meimpa and 40 mm 2-meimpg . although homopolymeric templates have been traditionally 
 used to 
 assess the reactivity of individual activated nucleotides , they are 
 not directly relevant to potentially functional sequences , which are 
 more likely to contain all four nucleotides . we therefore used a set 
 of mixed sequence templates to examine primer extension in a more 
 realistic setting . the template sequences followed the pattern 5-c5n-(primer binding sequence)-3 where n was a , u , su , or st . with the 5-c5a-(primer 
 binding sequence)-3 template , the corresponding rates of monomer 
 addition followed a similar pattern to those of the a6 template . the incorporation of either 2-meimpu , 2-meimpsu or 2-meimpst in the presence of 2-meimpg led to kmax values of 0.26  0.063 , 0.96  0.11 , and 4.4 
  0.45 h , respectively ( figure 2c , buffer 1 containing 200 mm hepes ph 7.0 , 200 mm mgcl2 and 1 m nacl ; also see supporting information 
 figure 1b(2 ) ) . similarly , we found that the incorporation of 
 2-meimpa in the presence of 2-meimpg was approximately an order of 
 magnitude faster on the single - u , su or st 
 templates than on the corresponding homopolymeric templates , with 
 observed rates of 0.83  0.045 , 0.38  0.019 , and 0.78  
 0.024 h , respectively ( figure 2d , buffer 1 ; see also supporting information 
 figure 1b(2 ) ) . however , as with the homopolymer templates , 
 thiolation of u or t in the template had little effect on the rate 
 of primer extension . on the basis of the fact that base stacking plays 
 a role in determining the melting temperature of complementary oligonucleotides , we suggest that the increased rates on the single - u 
 or a templates , compared to the homopolymer templates , are best explained 
 by improved stacking interactions of the incoming monomer ( adjacent 
 to the primer ) with downstream g monomers base paired to the c5 portion of the template . the above experiments were 
 carried out using a high salt buffer 
 ( buffer 1 , containing 200 mm mgcl2 and 1 m nacl ) that promotes 
 base pairing by masking the interstrand repulsion of negatively charged 
 phosphates . however , molar salt concentrations are incompatible with 
 primitive fatty acid based vesicles , and 
 we therefore examined primer extension without added nacl and with 
 a lower mgcl2 concentration . we observed that in buffer 
 2 ( 200 mm hepes ph 7.0 and 100 mm mgcl2 ) , kmax and kd values were modestly 
 reduced , by less than 2-fold in most cases ( table 1 ; figure 2c , d ; see also supporting information figure 1b(3 ) ) . we used next generation sequencing 
 ( ngs ) to assess the fidelity 
 of the primer extension products obtained on mixed sequence templates , 
 in the presence of competing activated monomers . we generated libraries 
 of extended primers flanked by the necessary adaptor sequences at 
 both the 5 and 3 ends , as illustrated in figure 3 . briefly , nonenzymatic primer extension was carried 
 out with a primer ( p2 ) that included the 5-adaptor sequence 
 necessary for on - bead amplification during sequencing , and also included 
 a 5-biotin so that the template strand could be removed by 
 a convenient bind and wash procedure using magnetic streptavidin beads . following template removal , the nonenzymatically extended primer molecules 
 were released from the streptavidin beads . an adaptor was then ligated 
 to the 3-end of each primer , followed by reverse - transcription 
 and pcr to yield the library for sequence analysis . ( a ) top : primer - extension was carried out on 2.0 m template 
 t9 ( agagag ) in the presence of 40 mm 2-meimpc and 50 mm 2-meimpu * 
 ( u * = u , su , or st ) on ice for 7 days . bottom : 
 products were sequenced , and the sequence reads binned according to 
 the number of nucleotides added to the primer ; y = c or u*. reaction 
 conditions : 2.0 m primer p2 , 200 mm hepes ph 7.0 , 100 mm mgcl2 . ( b ) top : primer - extension was carried out on 2.0 m 
 template t5 ( uc5 ) , t6 ( suc5 ) , or 
 t7 ( stc5 ) in the presence of 40 mm 2-meimpg 
 and 50 mm 2-meimpa on ice for 7 days . bottom : products were sequenced 
 and the sequence reads binned according to the number of nucleotides 
 added to the primer ; r = a or g. we compared primer extension across a 5-gagaga-(primer 
 binding sequence)-3 template ( t9 ) with u , su , 
 and st as activated monomers ( figure 4a ; see also supporting information table 
 1a ) in the presence of 2-meimpc and sequenced the extended 
 primers as described above to assess the extent and fidelity of the 
 template copying reaction . primer extension with 2-meimpu and 2-meimpc 
 yielded largely products extended by only two or three nucleotides 
 ( figure 4a , left panel ) . however , in an otherwise 
 identical reaction with 2-meimpsu and 2-meimpc we observed 
 a wider distribution of product lengths including full - length product , 
 i.e. , primer + six nucleotides ( figure 4a , 
 middle panel ) . this effect was further enhanced when 2-meimpst replaced 2-meimpu ( figure 4a , right 
 panel ) . sequence reads obtained 
 from the products of primer - extension on template t9 ( agagag ) , as 
 described in figure 4 , were sorted into bins 
 according to the number and identity of nucleotides added to the primer . ( a ) left : products extended by at least two nucleotides , with correct 
 incorporation of u * at position 1 , were sorted according to whether 
 the correct nucleotide c or the incorrect nucleotide u * was incorporated 
 at position 2 . right : products extended by at least two nucleotides , 
 with incorrect incorporation of c at position 1 , were sorted according 
 to whether the correct nucleotide c or the incorrect nucleotide u * 
 was incorporated at position 2 . ( b ) left : products extended by at 
 least three nucleotides , with correct incorporation at positions 1 
 and 2 , were sorted according to whether the correct nucleotide u * 
 or the incorrect nucleotide c was incorporated at position 3 . right : 
 products extended by at least three nucleotides , with incorrect incorporation 
 of u * at position 2 , were sorted according to whether the correct 
 nucleotide u*or the incorrect nucleotide c was incorporated at position 
 3 . examination of the sequences of the products of 
 primer extension 
 on the 5-gagaga-(primer binding sequence)-3 template 
 revealed enhanced fidelity with the 2-meimpsu and 2-meimpst monomers , compared to the standard 2-meimpu monomer . in 
 order to examine the effect of the modified nucleotides on g : u wobble 
 mispairing , we examined all products in which the primer had extended 
 by two or more bases and where the first nucleotide added was the 
 correct u ( or su or st ) . correct primer extension 
 would then result in incorporation of a c at position 2 , while g : u 
 mispairing would result in the incorporation of a u ( or su or st ) . we found that g : u mispairing at the second 
 position was diminished from 4.3% in the case of 2-meimpu to 1.6% 
 with 2-meimpsu and 2.0% with 2-meimpst ( figure 5a ; see also supporting information 
 table 2 ) , corresponding to a 23 fold improvement in 
 fidelity with the 2-thiolated u or t monomers . because primer extension 
 with 2-meimpu was so inefficient , we were unable to compare the frequency 
 of watson crick vs wobble pairing at the next g residue in 
 the template , which is at position 4 . surprisingly , we discovered 
 that a : c mispairing was also diminished 
 when u was replaced with su or st . the expected 
 product of primer extension on the 5-gagaga-(primer binding 
 sequence)-3 template at positions 1 , 2 , and 3 was primer - u*cu*. 
 when we examined sequences where the first two nucleotides were correct , 
 we found that an incorrect c was incorporated at position 3 over 19% 
 of the time , following primer extension with 2-meimpu and 2-meimpc . we noted a 56 fold drop in the amount of primer - ucc when 2-thiolated 
 u - derivatives were used , with only 4.3% and 2.9% c incorporation at 
 position 3 when primer extension was carried out with 2-meimpsu and 2-meimpsu , respectively ( figure 5b ; see also supporting information 
 table 2 ) . finally , we observed that both g : u and a : c 
 misincorporation largely 
 terminated further primer extension and , furthermore , that the small 
 amount of continued primer extension was highly error prone . we first 
 consider the case of a g : u mismatch at position 2 , corresponding to 
 primer - uu products ( see supporting information 
 table 2 ) . primer extension in the presence of 2-meimpu and 
 2-meimpc resulted in 2394 such sequences , but only 21 sequences corresponding 
 to the addition of one more nucleotide to the growing primer , i.e. , 
 less than 1% continued extension . in contrast , we observed 53  134 
 sequences corresponding to the correct 2-nucleotide extension product 
 primer - uc , and 13  077 sequences corresponding to continued 
 primer extension by at least one more nucleotide , i.e. , about 20% 
 continued primer extension . remarkably , primer extension following 
 a g : u mismatch was highly error prone , with an error rate of almost 
 40% . replacing 2-meimpu with either 2-meimpsu or 2-meimpst did not significantly increase the fidelity of post mismatch 
 synthesis ( 2550% error rate in both cases ) . examination of 
 sequences corresponding to an a : c mismatch at position + 1 reveals 
 similar poor fidelity at the following position . to assess the 
 capacity of su and st in 
 the template to mitigate g : u mispairing , we performed nonenzymatic 
 primer extension reactions with 5-c5u*-3 
 templates ( where u * = u , su , or st ) in the 
 presence of both 2-meimpa and 2-meimpg . interestingly , there was insignificant 
 differentiation between u , su , or st when 
 they were templating primer extension in this context , and the distributions 
 of product length were comparable in all three cases ( figure 4b ; see also supporting information 
 table 1b ) . a single - atom substitution 
 of oxygen by sulfur at the 2-position 
 of the activated uridine nucleotide 2-meimpu significantly improves 
 the rate and fidelity of nonenzymatic template - directed primer extension . 
 under different template and buffer conditions , the rate of primer 
 extension consistently followed the order st > su > u. surprisingly , this enhanced rate and fidelity of 
 template 
 copying was only observed with 2-thio - u or -t as the activated monomer , 
 and no significant effect was observed when these modified nucleotides 
 were present in the template strand . although the reasons for this 
 striking difference are not entirely clear , we suggest the following 
 speculative explanation . we consider first an incoming u ( or su or st ) monomer . although an su : a 
 base pair at an internal position in a duplex stem is much more stable 
 than a standard u : a base pair , little if any stabilization is seen 
 for an su : a base pair at the end of a helix . this is consistent with the fact that we do 
 not see a statistically significant decrease in the kd of the 2-meimpsu or 2-meimpst monomers relative to 2-meimpu ( although the kd values for the very slow reactions on the a6 template 
 have large standard errors ) . we do however see an increased maximal 
 rate of reaction ( at saturating monomer concentration ) for the 2-thiolated 
 monomers . we suggest that this stems from preorganization of the 2-thionucleotides 
 in the 3-endo conformation , which could 
 help to properly position and orient the phosphate group of the incoming 
 monomer for reaction with the attacking 3-hydroxyl of the 
 primer . weaker hydrogen bonding between the sulfur of su(t ) and the imino proton of g would weaken wobble pairing , favoring 
 correct binding of c , and that together with a further distorted geometry 
 may account for the decreased formation of u : g mismatches observed 
 with su and st . we attribute the decreased 
 frequency of a : c mismatches to the faster reaction rate of su or st ( relative to u ) when paired with a ; effectively 
 su and st outcompete c so that a : c mismatches 
 do not have time to form . we now consider the substitution of u in 
 the template with su or st . in this case , 
 the binding of an incoming a monomer to the primer template 
 duplex is facilitated by the stronger stacking interactions of the 
 purine nucleobase with flanking nucleotides , i.e. , the 3-nucleotide 
 of the primer , and downstream monomers . as a result , the kd of activated a is lower than that of the activated u 
 monomers . in addition , template preorganization by an internal su or st may contribute to enhanced binding of 
 2-meimpa , as observed for the 5-cccccu * templates . however , 
 once the activated a monomer is bound to the template , it has the 
 same reactivity whether the template base is u , su , or 
 st , and as a result , the rate at saturation ( i.e. , kmax ) does not change . accurate direct measurements 
 of binding affinities and geometries may allow for experimental testing 
 of the above hypotheses . the stalling of primer extension following 
 a mismatch has previously 
 been shown , under certain circumstances , to lead to an enhanced effective 
 fidelity of replication , because the first template copies to be completed 
 tend to be the most accurate . however , 
 this effect comes at the cost of significantly slowed overall rate 
 of replication . our data suggest that the formation of both u : g and 
 c : a wobble pairs during nonenzymatic template - directed primer extension 
 leads to a very strong stalling effect , i.e. , a greatly decreased 
 rate of primer extension following a wobble mismatch . the magnitude 
 of this effect ( ca . 20-fold , figure 5 ) is surprising 
 and should be examined in additional sequence contexts . nevertheless , 
 we now expect that replacing u with su or st should improve the overall rate of primer extension not only because 
 of the increased rate at which the 2-thio monomers are incorporated 
 but also because the increased accuracy of primer extension will lead 
 to less stalling after mismatches . in addition , we note that the strongly 
 decreased fidelity that we observed for primer extension following 
 a mismatch is consistent with previous proposals that errors introduced 
 during nonenzymatic rna copying may be dominated by multiple sequential 
 errors , as opposed to isolated single mutations . such clustered mutations may speed the exploration of sequence 
 space and the optimization of functions under selection . enhancing 
 the ability of rna to make large jumps through sequence space may 
 be particularly important in facilitating the emergence of novel rna - coded 
 functions . finally , our results raise the question of the prebiotic 
 availability 
 of su or st . it is conceivable that these 
 2-thio nucleotides could be generated spontaneously in a sulfur - rich 
 early earth environment through a route analogous to that proposed 
 for the synthesis of the canonical pyrimidine nucleotides . the experimental demonstration of an efficient 
 pathway for the prebiotic synthesis of either su or st nucleotides would support their proposed role in facilitating 
 nonenzymatic rna replication during the origin of life . representative 
 reaction 
 protocol : 4.0 l of 1.0 m hepes ph 7.0 buffer , 5.0 l 
 of nuclease - free water , 1.0 l of primer p1 , and 3.0 l 
 of template t5 were combined in a thin - walled pcr microtube . after 
 being mixed well by pipetting up and down multiple times , the oligonucleotides 
 were incubated at 90 c for 5 min and annealed at 25 c 
 for 5 min . a 2.0 l amount of 1.0 m mgcl2 was then 
 added to the solution and mixed well . to initiate primer extension , 
 1.0 l of 1.0 m 2-meimpa and 4.0 l of 200 mm 2-meimpg 
 were added to the lid of the microtube cap . the reaction was initiated 
 when the nucleotides were spun down into the buffered primer / template 
 solution . the solution was mixed well and incubated in a metal block 
 in an ice bath for the duration of the experiment . aliquots ( 4.0 l 
 each ) were removed at 10 , 20 , 30 , 40 , and 50 min and were immediately 
 quenched by addition to 26 l of precipitation buffer [ 3.0 l 
 of 3.0 m naoac ph 5.5 , 2.0 l of 500 mm edta ph 8.0 , 1.0 l 
 of 5 mg / ml glycogen , 2.0 l of 10x tbe ( 1.0 m tris , 1.0 m boric 
 acid , and 20 mm edta ph 8.0 ) , and 18.0 l 8.0 m urea ] . after 
 the mixture was briefly vortexed to mix the contents , 75 l 
 of pure ethanol was added . the sample was mixed and kept at 25 
 c for a minimum of 30 min and was then centrifuged at 15  000 
 rcf in an eppendorf 5424r centrifuge at 4 c . the rna pellets 
 were then taken up in 5.0 l of 8.0 m urea in 1x tbe and incubated 
 at 90 c for 5 min before the rna products were separated by 
 20% ( 19:1 ) denaturing page . the gel was scanned with a typhoon 9410 
 variable mode imager , and the bands were quantified using the accompanying 
 imagequant tl software package . nonenzymatic template - directed 
 primer extension reactions were performed as described above except 
 that 2.0 m primer p2 and 2.0 m template were used . ten millimolar tris - hcl ( ph 7.5 ) , 
 10 mm edta ( ph 8.0 ) , 2.0 m nacl ; buffer a : 100 mm naoh , 5 mm nacl ; 
 buffer b : 10 mm nacl ; elution buffer : 95% formamide , 10 mm edta ( ph 
 8.0 ) ; ssc buffer : 150 mm nacl , 15 mm na citrate , ph 7.0 . a 100 l amount of streptavidin 
 myone c1 dynabeads ( 10 mg / ml ; invitrogen ) was separately decanted 
 for each library assembly reaction . gentle draw / expel pipetting was 
 used as a bead mixing technique ; vortexing was avoided . the beads 
 were mixed and washed 3 with 200 l  bind and wash  
 buffer and were allowed to sit on a magnetic rack for 1 min following 
 each wash step . beads were then washed 2  200 
 l buffer a , followed by 2  200 l buffer b. the 
 rna pellet from an extension reaction using primer p2 was diluted 
 with 200 l of nuclease - free water and 200 l of  bind 
 and wash  buffer . the rna solution was added to the washed 
 and decanted beads and then briefly mixed to suspend the beads in 
 the rna solution . after the mixture was rested on the magnetic rack for 1 min , the supernatant 
 was drawn off . beads with 
 bound primer / template complex were mixed with 250 l of ssc 
 buffer and decanted after resting on the magnetic rack for 1 min . the beads were suspended in 100 l of 150 mm naoh and incubated 
 at 25 c for 10 min . the tube was placed on the magnetic rack 
 for 1 min , and the supernatant was drawn off . the beads were then 
 mixed with 250 l of 100 mm naoh and immediately placed on the 
 magnetic rack for 1 min followed by removal of the supernatant . the 
 biotinylated oligonucleotides were immediately eluted from the beads 
 by mixing with 250 l of elution buffer at 65 c for 5 
 min followed by 1 min on the magnetic rack and removal of the supernatant . the rna from this final supernatant was precipitated upon addition 
 of 1120 l of ethanol and 30 l of 3.0 m naoac ph 5.5 
 and incubation for 30 min at 25 c . a 20.0 l 
 amount of 100 m 3-adaptor ( 5-phosphate - aga 
 tcg gaa gag cac acg tct3-3-t-5 ; dna ) and 6.0 
 l of nuclease - free water were added to the biotinylated rna 
 pellet and placed in a thin - walled pcr microtube . after dissolution , 
 the mixture was incubated at 65 c for 2 min then placed on ice , 
 and 4.0 l of 10x t4 rna ligase i buffer , 4.0 l of 50% 
 peg 8000 , 4.0 l of dmso ( molecular biology grade ) , and 2.0 
 l of t4 rna ligase i ( 10,000 u / ml ) were added . after mixing , the solution was incubated at 37 c for 2 h. the samples were 
 cleaned up with a qiaquick pcr purification kit ( eluted with h2o not eb buffer from kit ; qiagen ) to remove excess 3-adaptor 
 ( 22-mer ) and retain the ligated product ( 92-mer ) . the samples were 
 then lyophilized to dryness and taken up in 15 l of nuclease - free 
 water . the following sequences 
 used a combination of materials from superscript iii first - strand 
 synthesis supermix ( invitrogen ) and nebnext multiplex small rna library 
 prep set 1 for illumina ( neb ) . a 1.0 l amount of sr rt primer 
 for illumina ( diluted 1:2 ; neb ) and 2.0 l of annealing buffer 
 ( invitrogen ) were added to the 15 l solution of rna . the mixture 
 was incubated in a thin - walled pcr microtube at 75 c using a 
 thermal cycler for 5 min , 37 c for 15 min , and finally 25 c 
 for 15 min . at 25 c , 20 l of 2x first - strand reaction 
 mix ( invitrogen ) and 2.0 l of superscript iii / rnaseout enzyme 
 mix ( invitrogen ) were added to the rna solution , mixed , and incubated 
 at 50 c for 1 h and 85 c for 5 min and then kept on ice . 
 a 2.5 l amount of sr primer for illumina ( neb ) , 2.5 l 
 of indexed prime ( neb ) , 5.0 l of nuclease - free water , and 50 
 l of longamp taq 2x master mix ( neb ) were 
 added to the reverse - transcribed mixture and were cycled 12 times 
 ( 30 s of initial denaturation , 94 c ; 15 s , 94 c ; 30 s , 
 62 c ; 15 s , 70 c ; 5 min of final extension at 70 c ; 
 hold at 4 c ) . the desalted and 
 enzyme - free dna was purified on a 20% tbe precast gel cassette ( invitrogen ) 
 with quick - load pbr322 dna - mspi digest ( neb ) as a marker . the target 
 band of 140 bp was sliced out , crushed with a disposable plastic rnase - free 
 pestle ( fisher ) , and eluted with 250 l of dna gel elution buffer 
 ( neb ) . the dsdna was desalted with a qiaquick pcr purification kit 
 and quantified by qpcr with a kapa sybr fast universal qpcr kit for 
 illumina ( kapa biosystems ) . samples were prepared as per the 
 manufacturer s recommendations with a 30% phix spike to bring 
 an appropriate level of initial sequence diversity to the libraries . a multiplexed paired - end protocol was used : 25 nucleotides for read-1 , 
 index read , 25 nucleotides for read-2 . upon completion of sequencing the 
 script takes two fastq files , one for the forward read and one for 
 the reverse read , and filters out reads that either lack the exact 
 adapter sequence in the reverse read , lack the adapter in the forward 
 read ( with a maximum edit distance tolerance of 1 ) , or have forward 
 and reverse reads that are not identical in the region of nonenzymatic 
 primer extension . it then outputs all retained sequences and their 
 corresponding total read counts , as well as the number of reads discarded 
 for the reasons mentioned above .