Optimized protocol for quantifying 5′ UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting

Summary Direct analysis of ribosome targeting (DART) allows investigators to measure the translation initiation potential of thousands of RNAs in parallel. Here, we describe an optimized protocol for generating active translation extract from S. cerevisiae, followed by in vitro translation, purification of ribosome-bound RNAs, and subsequent library preparation and sequencing. This protocol can be applied to a variety of cell types and will enable high-throughput interrogation of translational determinants. For complete details on the use and execution of this protocol, please refer to Niederer et al. (2022).1

SUMMARY Direct analysis of ribosome targeting (DART) allows investigators to measure the translation initiation potential of thousands of RNAs in parallel. Here, we describe an optimized protocol for generating active translation extract from S. cerevisiae, followed by in vitro translation, purification of ribosome-bound RNAs, and subsequent library preparation and sequencing. This protocol can be applied to a variety of cell types and will enable high-throughput interrogation of translational determinants. For complete details on the use and execution of this protocol, please refer to Niederer et al. (2022). 1

BEFORE YOU BEGIN Preparation of yeast translation extracts
Timing: 2 days 1. Inoculate a starter culture by scooping a single yeast colony from a streak plate into 6 mL of PAD media. Incubate at 30 C on a shaker for 12-14 h. 2. Dilute the starter culture 1:20 and measure absorbance at 600 nm (OD 600 ).
a. Calculate the required volume of starter culture to inoculate 2 750 mL yeast cultures to achieve a final OD 600 of 12-15 in the desired length of time given the wild-type doubling time of approximately 90 min.
Note: We generally inoculate 750 mL cultures the night before lysate preparation, targeting the desired OD 600 of 12-15 the following morning (approximately 12 h of growth).
CRITICAL: Do not dilute the yeast below an OD 600 of 0.004 as the yeast will not recover consistently.
Note: The OD 600 values described here should be taken only as general guidelines. Cells should be harvested in late exponential growth phase to yield active lysate. OD 600 measurements can vary significantly between instruments and both the doubling time and OD 600 at which exponential growth occurs differs between strains. We recommend performing growth curve measurements to determine this empirically before proceeding to lysate preparation.
8. Weigh the 50 mL tube containing the yeast pellet and subtract the weight of the empty tube to determine the mass of the cell pellet. 9. Transfer a volume of Buffer A + M equal to at least 1.5 times the pellet mass, rounding up to the nearest 10 mL (i.e., 20 mL for a 10 g pellet) to a new 50 mL conical tube. a. Add 1 cOmplete Mini protease inhibitor tablet per 10 mL of Buffer A + M and dissolve. 10. Resuspend the cell pellet in a volume 1.5 times the pellet mass (i.e., 15 mL for a 10 g pellet) of Buffer A + M. 11. Add a mass of 0.5 mm zirconia/silica beads, pre-chilled to À20 C, equal to 5 times the mass of the cell pellet to the 50 mL tube. 12. Lyse the cells by vortexing for 1 min at maximum speed. a. Repeat this 5 times (for a total of 6 rounds) with 1 min of cooling on ice between vortexing to avoid heating of the solution. 13. Pellet the cell debris by centrifuging at 2,600 3 g for 5 min at 4 C. Transfer the supernatant to 50 mL Oak Ridge tubes. 14. Place the Oak Ridge tubes in an SS-34 or other compatible rotor and clarify by centrifuging at 26,700 3 g for 35 min at 4 C. 15. Transfer the supernatant to new Oak Ridge tubes and centrifuge again at 26,700 3 g for 12 min at 4 C. 16. While samples are centrifuging, hydrate a dialysis cassette in a beaker containing 2 L of Buffer A for 2 min at 4 C. 17. Carefully remove the supernatant, avoiding the bottom residual pellet and top lipid layer, and transfer it to a new 15 mL tube on ice. 18. Transfer the clarified extract into a 20 mL syringe. 19. Affix an 18-gauge needle and inject the extract into the hydrated dialysis cassette, carefully avoiding puncturing the cassette membrane. 20. Place the cassette into the beaker containing 2 L of Buffer A and dialyze for 45 min at 4 C.
a. Replace the buffer with 2 L of fresh Buffer A and continue dialysis for another 45 min at 4 C. 21. Collect the dialyzed extract from the cassette and aliquot into pre-chilled 1.5 mL tubes. Flash freeze in liquid nitrogen and store at À80 C.
23. Resuspend 200 mg of creatine phosphokinase powder in 10 mL of creatine phosphokinase resuspension buffer (20 mg / mL creatine phosphokinase). 24. Incubate the solution at 26 C for 30 min to reactivate the creatine phosphokinase. 25. Add 10 mL of 100% glycerol and mix well. The final 10 mg / mL creatine phosphokinase solution can be aliquoted and stored at À20 C for 6 months.

Pool design
Design a pool of DNA oligonucleotide sequences corresponding to the 5 0 UTR RNA sequences of interest. The pool used in our original 1 study consisted of up to 122 nucleotides of 5 0 UTR sequence and at least 24 nucleotides of coding sequence to cover the binding footprint of initiating ribosomes. 2 Append the T7 promoter sequence to the 5 0 end and a common primer sequence (key resources table) to the 3 0 end of the oligos. These sequences provide common handles for PCR amplification, in vitro transcription, and reverse transcription of the oligo pool. A 10-nucleotide unique barcode may also be included at the 3 0 end of the oligo prior to the common primer sequence to facilitate assignment of highly similar sequences. A schematic illustrating the oligo design is shown in Figure 1. Note that it is essential to remove any upstream start codons located upstream of the desired initiation codon. This is required to ensure that each RNA molecule can only recruit a single ribosome and to unambiguously assign a ribosome recruitment score to a given initiation context. In our original study, 1 upstream ATGs were changed to AGT.
Note: The oligonucleotide pool used in our original study 1 contained 12,000 unique 5 0 UTR sequences, and we have since performed DART analysis successfully on pools consisting of 24,000 unique sequences. Storage conditions: Store at À20 C for 6 months.

OPEN ACCESS
Note: Add RNasin Plus immediately before use.
Storage conditions: Buffer without RNasin Plus added may be stored at 25 C for 1 year.

Cycloheximide (10 mg / mL) solution
Dissolve 50 mg of cycloheximide powder in 5 mL of nuclease-free water.
Storage conditions: Prepare fresh before use.

cOmplete mini solution
Dissolve one cOmplete mini protease inhibitor tablet in 2 mL of nuclease-free water.
Storage conditions: Prepare fresh before use. Note: An example pool PCR optimization gel is shown in Figure 2.

RNA elution buffer
CRITICAL: The use of blue light illumination is necessary to prevent DNA damage that can result from UV illumination.
e. Excise PCR products with a razor blade and place gel slices into two 1.5 mL tubes containing 750 mL of DNA elution buffer. i. Rotate for 12-14 h at 25 C. f. Transfer the eluted DNA solution to new 1.5 mL tubes.
i. Add 750 mL of isopropanol and 2 mL of Glycoblue to each tube.
ii. Vortex the sample and incubate at À20 C for at least 30 min.
iii. Centrifuge at 21,000 3 g at 4 C for 30 min to pellet DNA.
Pause point: The purified DNA template can be stored at À20 C in isopropanol until ready for use.
g. Wash pellets with 750 mL of 70% ethanol and centrifuge at 21,000 3 g at 4 C for 10 min.
i. Remove supernatant and air-dry pellets at 25 C for 5 min.
ii. Resuspend the precipitated DNA in 8 mL of nuclease-free water.
Transcribing, capping, and biotinylating the RNA pool

Timing: 2-3 days
This step generates the RNA pool to be used in the ribosome recruitment assay. Addition of the 5 0 methylguanosine cap is required to emulate physiological cap-dependent translation. 3 0 biotinylation ensures efficient RNA recovery after incubation in translation extract and fractionation. Note: An example in vitro transcription product gel is shown in Figure 3.
CRITICAL: The use of blue light illumination is necessary to prevent RNA damage that can result from UV illumination.
f. Excise RNA bands using a razor blade and place gel slices into a 1.5 mL tube containing 750 mL of RNA elution buffer. i. Rotate for 12-14 h at 4 C. g. Transfer the eluted RNA solution to a new 1.5 mL tube for precipitation.
i. Add 750 mL of isopropanol and 2 mL of Glycoblue.
ii. Vortex the sample and incubate at À20 C for at least 30 min. Centrifuge at 21,000 3 g at 4 C for 30 min to pellet RNA.
Pause point: The purified RNA can be stored at À20 C in isopropanol until ready for use.

Reagent Amount
Transcription buffer (103)   iii. Wash pellets with 750 mL of 80% ethanol and centrifuge at 21,000 3 g at 4 C for 10 min. iv. Remove supernatant and air-dry pellets at 25 C for 5 min. v. Resuspend the precipitated RNA in 50 mL of nuclease-free water. h. Transfer 0.5 mL of RNA to a tube containing 9.5 mL of water. Load 2 mL of diluted RNA onto a Nanodrop and measure concentration, targeting approximately 40 micrograms total. 9. Add 5 0 methylguanosine cap as follows: a. Add 2 mL of RNasin Plus to the 50 mL of purified RNA. Heat-denature by incubating at 65 C for 5 min. Transfer denatured RNA to ice for 2 min. b. Prepare the capping reaction using the Vaccinia capping system (NEB).
c. Incubate the capping reaction at 37 C for 1 h. 10. Clean-up the capping reaction.
CRITICAL: Keep the samples on ice for the duration of the reaction clean-up.
a. Add 560 mL of binding buffer and 640 mL of isopropanol to the capping reaction. b. Apply 800 mL (maximum loading capacity) of the RNA solution to a Zymo V column. Centrifuge at 20,000 3 g for 1 min at 4 C. Repeat with the remaining 480 mL. c. Wash the column by adding 200 mL of 80% ethanol, then centrifuging at 20,000 3 g for 1 min at 4 C. d. Repeat the wash step twice more (for a total of three washes). e. To elute the RNA, add 200 mL of nuclease-free water and spin at 21,000 3 g for 2 min at 4 C. f. Precipitate the eluted RNA by adding 22 mL of 3 M sodium acetate, pH 5.3 and 1 mL of 100% ethanol. i. Incubate at À20 C for 30 min. Centrifuge at 21,000 3 g at 4 C for 30 min to pellet the RNA. g. Wash pellets with 1 mL of 80% ethanol and centrifuge at 21,000 3 g at 4 C for 10 min.
i. Remove supernatant and air-dry pellets at 25 C for 5 min.
ii. Resuspend the precipitated RNA in 6 mL of nuclease-free water. h. Transfer 0.5 mL of capped RNA to a tube containing 9.5 mL of water for concentration measurement. i. Load 2 mL of diluted RNA onto a Nanodrop and measure the capped RNA concentration.  ii. Determine the molarity of the capped RNA solution using the following approximate molecular weight calculation: Molecular Weight of ssRNA = (number of nucleotides 3 320.5) + 159.0. 11. Transfer 50 pmol of capped RNA to a new 0.2 mL tube. Add water to bring the total volume to 6 mL. a. Add 2 mL of DMSO. Heat-denature the RNA by incubating at 85 C for 4 min. b. Prepare the 3 0 biotinylation reaction using the Pierce 3 0 biotinylation kit as follows: 12. Incubate at 16 C for 12-14 h in a thermocycler. 13. Clean-up the biotinylation reaction. a. Add 70 mL of water and transfer reaction to 1.5 mL MaXtract tube. i. Add 100 mL of phenol:chloroform:IAA, 25:24:1, pH 6.6. ii. Vortex briefly and centrifuge at 16,000 3 g for 10 min at 4 C to separate the phases. b. Carefully transfer the top aqueous layer to a new 1.5 mL tube.
ii. Incubate at À20 C for at least 30 min to precipitate RNA.
iii. Centrifuge at 21,000 3 g at 4 C for 30 min to pellet the RNA. c. Wash pellets with 1 mL of 80% ethanol and centrifuge at 21,000 3 g at 4 C for 10 min. Remove supernatant and repeat for a second wash. d. Remove the supernatant and air-dry pellets at 25 C for 5 min. e. Resuspend the precipitated RNA in 1.2 mL of nuclease-free water.

Yeast extract preparation and ribosome recruitment
Timing: 0.5-1 h The yeast translation extract is first treated with micrococcal nuclease to degrade endogenous mRNA, increasing ribosome recruitment to the pool RNA. The ribosome recruitment reactions are carried out in the presence of cycloheximide to ensure that each RNA is only able to recruit a single ribosome that is then trapped on that RNA.
14. Degrade endogenous mRNA from the yeast translation extract by treating with micrococcal nuclease (MNase): a. Add 42 mL of 40 mM CaCl 2 to 3.5 mL of yeast translation extract.
CRITICAL: Supplementing the extract with CaCl 2 is essential as MNase is a Ca 2+ -dependent enzyme. 15. Prepare the in vitro ribosome recruitment reaction. The amount shown for the 3.23 allows for 3 individual replicate reactions with additional volume to allow for pipetting error.
a. Split each reaction in half by transferring 810 mL to 2 1.5 mL tubes. b. Add 190 mL of the capped and biotinylated RNA pool (20 pmol of RNA per tube). c. Incubate the reaction for 30 min on a thermomixer set to 600 RPM and 26 C. Immediately place on ice. d. Transfer 200 mL of each reaction into a new 1.5 mL tube. Snap-freeze these samples in liquid nitrogen and store at À80 C for subsequent input library preparation.

Isolation of 80S-RNA complexes and RNA extraction
Timing: 8-10 h In this step, RNAs bound to an 80S ribosome are separated from unbound through sucrose gradient fractionation. Cycloheximide is maintained in the sucrose solutions to ensure 80S-RNA complexes are maintained during fractionation. Hot phenol plus SDS RNA extraction is used to ensure complete denaturation of the concentrated ribosomal RNA and proteins.
16. Before you begin, pre-chill SW28 rotor and swing buckets to 4 C. Turn on ultracentrifuge and allow it to reach 4 C. 17. Prepare the sucrose gradients. This is done using a Biocomp Gradient Master/Fractionator station and associated parts and accessories. a. Begin by preparing 10% and 50% sucrose solutions. b. Using the Marker Block, mark the long cap halfway point of Seton Polyclear ultracentrifuge tubes. c. Add the 10% sucrose solution until approximately 1 cm above the mark. d. Draw 60 mL of 50% sucrose solution into a 60 mL syringe. e. Affix a wide-bore pipetting needle to the syringe. f. Depress the plunger until the solution is at the end of the needle, then hold tension to prevent dripping. Insert the needle to the bottom of the tube containing 10% sucrose. g. Slowly dispense the 50% sucrose solution until the interface reaches the marked point. Hold tension on the syringe to prevent dripping and remove from the tube. i. Repeat the underlaying process for the remaining tubes. h. Place the long caps on the filled tubes, avoiding bubbles, and remove any excess solution from the top of the cap. i. Place the tubes in the Magnabase holder, then place the holder onto the Gradient Master mixing station. j. Select the pre-installed program for the SW28 10%-50% sucrose gradient (long) and run the gradient preparation. k. Place the prepared gradients in SW28 buckets and chill at 4 C for 30 min. 18. Slowly remove the caps and then gently load the 1.8 mL ribosome recruitment reaction onto the top of the gradient. 19. Precisely balance the swing buckets and spin the gradients for 3 h at 27,000 RPM (approximately 97,000 3 g) at 4 C. 20. Fractionate the sucrose gradients using a Gradient Master with a fraction collector attached.
Note: An example gradient trace is shown in

Timing: 1.5 days
The RNA extracted from the 80S fraction contains a large amount of contaminating ribosomal RNA. In this step, the biotinylated RNA pool is pulled down with streptavidin beads to remove this ll OPEN ACCESS contamination from subsequent library preparation steps. The reserved RNA pool and input samples are also pulled down to account for potential biases in pull-down efficiency.
42. Briefly vortex the magnetic streptavidin beads to resuspend. Transfer 125 mL of bead slurry per sample to a LoBind 1.5 mL tube. 43. Apply the tubes to a magnetic rack to separate the beads for 30 s. Remove the supernatant.
Equilibrate the beads by washing the beads twice with 100 mL of streptavidin binding buffer. 44. Denature the RNA samples by incubating at 65 C for 5 min. Transfer to ice for 3 min. 45. Transfer the RNA samples to the tubes containing the magnetic beads. Vortex briefly to mix.
Incubate the samples for 12-14 h at 4 C, rotating. 46. Apply the tubes to the magnetic rack and allow to separate for 2 min. Remove the supernatant.
Wash the samples three times with 1 mL of streptavidin wash buffer. 47. To elute, resuspend the beads in 50 mL of streptavidin elution buffer. Incubate at 70 C for 5 min. 48. Apply the tubes to the magnetic rack and transfer the eluted RNA to new 1.5 mL tubes. 49. Add 400 mL of water, 50 mL of 3 M sodium acetate pH 5.3, 2 mL of Glycoblue, and 600 mL of isopropanol. Incubate the samples at À20 C for at least 30 min to precipitate RNA.
Pause point: The purified RNA can be stored at À20 C in isopropanol until ready for use.
50. Centrifuge at 21,000 3 g at 4 C for 30 min to pellet the RNA. 51. Wash pellets twice with 1 mL of 80% ethanol and centrifuge at 21,000 3 g at 4 C for 10 min. 52. Remove supernatant and air-dry pellets at 25 C for 5 min. 53. Resuspend the RNA in 11 mL of water and proceed immediately to reverse transcription.

Reverse transcription and adaptor ligation
Timing: 2-3 days Here, the purified RNA is reverse transcribed into single-stranded cDNA. We gel-purify full-length cDNA to capture only the desired product for subsequent steps. An adaptor containing a common PCR handle and a 10N randomer unique molecular identifier (UMI) sequence for PCR duplicate collapsing is then ligated to the 3 0 end of the cDNA. The adaptor ligation reactions are then cleaned up using Silane beads.
54. Transfer the resuspended RNA to PCR tubes for RT primer annealing. a. Add 1 mL of 10 mM RT primer to the resuspended RNA. b. Transfer the samples to a thermocycler. Denature the RNA and anneal the RT primer with the following program: 55. Transfer the samples to ice. 56. Prepare the Superscript III reverse transcription master mix as follows: Note: The amount shown is for one reaction and should be scaled as appropriate.
Steps Temperature Time Note: An example cDNA gel is shown in Figure 5.
CRITICAL: The use of blue light illumination is necessary to prevent DNA damage that can result from UV illumination.
63. Excise cDNA synthesis products with a razor blade and place gel slices into two 1.5 mL tubes containing 750 mL of DNA elution buffer. 64. Rotate the samples for 12-14 h at 25 C. 65. Transfer the eluted DNA solution to new 1.5 mL tubes. 66. Add 750 mL of isopropanol and 2 mL of Glycoblue to each tube. Vortex the sample and incubate at À20 C for at least 30 min. 67. Centrifuge at 21,000 3 g at 4 C for 30 min to pellet DNA. 68. Wash pellets twice with 1 mL of 80% ethanol and centrifuge at 21,000 3 g at 4 C for 10 min.
Remove supernatant and air-dry pellets at 25 C for 5 min.
Pause point: The purified cDNA can be stored at À20 C in isopropanol until ready for use. These steps prepare next-generation sequencing libraries from the adaptor-ligated cDNA samples. The PCR primers used provide Illumina adaptor sequences with the reverse primer including a sample-specific 6-nucleotide barcode for sample identification. In the first step, we run small-scale diagnostic PCRs across a range of PCR cycles to identify the optimal cycle number for library construction without producing spurious overamplification products and large numbers of PCR duplicates. With the optimal cycle number identified, we run full-scale library construction PCRs and gel-purify the library products. The libraries are then quantified on Qubit and run on a Bioanalyzer to check library purity.
76. Prepare the diagnostic PCR reactions master mix.
Note: The amount shown is sufficient for 30 diagnostic reactions: Note: An example diagnostic PCR gel is shown in Figure 6.
CRITICAL: The use of blue light illumination is necessary to prevent DNA damage that can result from UV illumination.

81.
With the optimal PCR conditions determined, prepare the final PCR reaction master mix.  Note: An example final PCR gel is shown in Figure 7.
CRITICAL: The use of blue light illumination is necessary to prevent DNA damage that can result from UV illumination.
92. Excise the library products with a razor blade and place gel slices into two 1.5 mL tubes containing 750 mL of DNA elution buffer. 93. Incubate on a rotator for 12-14 h at 25 C. Remove supernatant and air-dry pellets at 25 C for 5 min. 98. Resuspend the DNA library in 10 mL of water. 99. Run 1 mL of each sample on an Agilent Bioanalyzer using a High Sensitivity DNA chip to determine the library size distribution.
Note: A successful library preparation should yield a single dominant peak of the appropriate size. An example size distribution is shown in Figure 8.
100. Pool the samples into a single multiplexed library at a final concentration of 1-2 nM each.
Note: To obtain sufficient coverage for a 12,000-sequence pool, we used one lane of Illumina HiSeq 2500 sequencer to obtain 150 million 75 bp paired end reads.

EXPECTED OUTCOMES
A successful outcome is shown in Figure 5 (RT gel), 7, and 8. First, we expect to see a clear and defined RT product from the input lane ( Figure 5), though we do not always observe signal at this stage for the other samples. Relatedly, we expect to see products of the correct size at the final PCR stage (Figure 7), which should correspond to a high quality and narrow size distribution when visualized on a Bioanalyzer or other similar quality control instrument. Lastly, we expect greater than 75% mapping efficiency to the synthetic genome used in the experiment. Note:

QUANTIFICATION AND STATISTICAL ANALYSIS
The percentage of PCR duplicates should not exceed 30%. Usable data may still be obtained from libraries containing a higher percentage of duplicates, but this increases the number of wasted sequencing reads.

UMI removal
Next, we remove the UMI sequence from the collapsed reads.
Generate a STAR reference genome Before aligning the processed reads, create a reference genome to align to. The input for this genome generation is a fasta-formatted file containing the oligo sequences minus the T7 promoter, as this is not included in the transcribed RNA sequence. In this example, this input file is referred to as ''DARTref.fa''.
Aligning the reads with STAR With the reference genome created and the reads processed, align the reads using STAR.
Note: Reads that fail to align should be excluded from further analysis. A successful experiment and analysis should yield > 80% uniquely mapping reads.
Count the reads mapping to each oligo sequence Next, convert the raw read counts to counts per million (CPM) by dividing the number of reads per sequence by the total number of reads in that library (in millions).

Calculate ribosome recruitment scores (RRS)
The CPM values can now be used to calculate the RRS values for each sequence by taking the ratio of normalized 80S reads to normalized initial pool reads as follows: CPMðPoolÞ :

LIMITATIONS
Each extract preparation must be tested to determine the optimal RNA concentration for DART. We generally target high, but non-saturating concentrations as evaluated by luciferase assays. To achieve the desired 20 pmol per reaction this may require scaling up the size of the reaction, and therefore the amount of extract required. Lastly, any RNAs that may co-sediment with the 80S fraction as part of an unrelated large RNP will be identified as ribosome bound.

Problem 1
Difficulty with phenol/chloroform extraction or resuspension in step 41.

Potential solution
When reaction volumes are large (greater than 2 mLs) resuspension of the pellet can be challenging, presumably due to the large amount of ribosomal RNA in the samples. In this case an alternative is to forgo the phenol/chloroform extraction and precipitation altogether and instead run the combined fractions onto a desalting column to achieve the desired buffer conditions for streptavidin pulldown.

Problem 2
Low yields or broad size distribution of final library prep in step 99.

Potential solution
When fraction collection takes an extended amount of time the already collected samples may experience some degree of degradation. If this is a concern, fractions can be collected directly into phenol/chloroform solution to preserve the integrity of the samples.

Problem 3
Low yield in final library prep in step 99.

Potential solution
One of the main reasons for low yield is low input RNA followed by consistent loss of RNA throughout the procedure. To minimize loss during handling we extensively utilize GlycoBlue to ensure RNA pellets are not lost. If low yield continues to be an issue it is likely that the reaction volume needs to be scaled up to increase the amount of total input RNA from the beginning of the procedure.

Problem 4
High percentage of PCR duplicates in final library following step 100.

Potential solution
Decrease the number of PCR cycles used to generate the final library.
Problem 5 Smeared cDNA gel in step 62.

Potential solution
This often occurs as a result of incomplete RNA degradation. Increase the concentration of sodium hydroxide used and/or extend the incubation time to fully degrade RNA.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Wendy V. Gilbert (wendy.gilbert@yale.edu).

Materials availability
This study did not generate new unique reagents. Data and code availability All datasets associated with the original study 1 have been deposited at GEO and the accession numbers are available therein. This study did not generate original code.