TITLE: Massive Parallel Reporter Assay (MPRA)
AUTHORS: Gracie Gordon, Nadav Ahituv

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence, which is measured via RNA sequencing and normalized for cellular integration via DNA sequencing of the barcode. This technology has been used to test thousands of sequences and their variants for regulatory activity, to decipher the regulatory code and its evolution, and to develop genetic switches. Lentivirus-based MPRA (lentiMPRA) produces ‘in-genome’ readouts and enables the use of this technique in hard-to-transfect cells. Here, we provide a detailed protocol for lentiMPRA, along with a user-friendly Nextflow-based computational pipeline—MPRAflow—for quantifying CRS activity from different MPRA designs. The lentiMPRA protocol takes ~2 months, which includes sequencing turnaround time and data processing with MPRAflow.</div></div>

[STEPS]
?. [Library Amplification]
Set up the first-round PCR reaction. This reaction adds a vector overhang sequence upstream andminimal promoter and adaptor sequences downstream of the CRSs  ABC1ReagentVolume (μL)Final conc2Agilent oligonucleotide (100 nM)21nM3NEBNext High-Fidelity 2× PCR Master Mix1001x45BC-AG-f01 (100 μM)10.5uM55BC-AG-r01 (100 μM)10.5uM6Ultrapure distilled H2O967Total volume200
ABC1ReagentVolume (μL)Final conc2Agilent oligonucleotide (100 nM)21nM3NEBNext High-Fidelity 2× PCR Master Mix1001x45BC-AG-f01 (100 μM)10.5uM55BC-AG-r01 (100 μM)10.5uM6Ultrapure distilled H2O967Total volume200
?. [Library Amplification]
Split the premixture into five PCR tubes (40 μL per tube)CRITICAL STEP Splitting the PCR reaction into multiple tubes is important to reduce the risk ofPCR ‘jackpotting’ (errors that occur during the early PCR cycles and get amplified exponentially) oramplification bias accidentally occurring during PCR
?. [Library Amplification]
Run the PCR reaction as follows:  ABCD1Cycle No.DenatureAnnealExtend2198oC, 2min32-6 (5cycles)98oC, 15sec60oC, 20sec720C, 30sec4772oC, 5min
ABCD1Cycle No.DenatureAnnealExtend2198oC, 2min32-6 (5cycles)98oC, 15sec60oC, 20sec720C, 30sec4772oC, 5min
?. [Library Amplification]
Combine the PCR products in a DNA LoBind tube.
?. [Library Amplification]
Bring the HighPrep PCR reagent to RT for at least 30 min before use. Shake thoroughly to fully
resuspend the magnetic beads. Add 1 volume (200 μL) of HighPrep PCR reagent and mix
thoroughly by pipetting up and down 6–8 times
?. [Library Amplification]
Incubate the mixture for 5 min at RT.
?. [Library Amplification]
Place the tube on the magnet for 2–3 min until the solution clears and beads pull to the side ofthe wall
?. [Library Amplification]
Carefully remove the supernatant by pipetting without disturbing the beads. A small amount(10–20 μL) of supernatant can be left in the tube
?. [Library Amplification]
With the tube on the magnet, add 500 μL of 80% (vol/vol) ethanol and incubate for 30 s.
?. [Library Amplification]
Remove the ethanol by pipetting while the tube is still on the magnet.
?. [Library Amplification]
Repeat the 80% (vol/vol) ethanol washing (Steps 10 and 11)
?. [Library Amplification]
Flash-spin (200–1,000g, 22–25 °C, 3 s) the tube, immediately place it back on the magnet andremove the supernatant
?. [Library Amplification]
Dry the bead pellet for 2–3 min. Do not overdry the beads.
?. [Library Amplification]
Add 50 μL of Buffer EB to the beads and mix by pipetting and vortexing.
?. [Library Amplification]
Place the tube on the magnet for 1–2 min until the solution is clear.
?. [Library Amplification]
Transfer 45–50 μL of the eluate to a DNA LoBind tube.
?. [Library Amplification]
Measure the DNA concentration using a NanoDrop spectrophotometer. The expected concentrationis 5–20 ng/μL.
?. [Library Amplification]
Set up the second-round PCR reaction. This reaction adds a 15-bp barcode and vector overhangsequence downstream of the first-round PCR fragment  ABC1ReagentVolume (μL)Final conc2First-round PCR productVariable (100ng)1nM3NEBNext High-Fidelity 2× PCR Master Mix2001x45BC-AG-f01 (100 μM)20.5uM55BC-AG-r01 (100 μM)20.5uM6Ultrapure distilled H2OUp to 400uL7Total volume400
ABC1ReagentVolume (μL)Final conc2First-round PCR productVariable (100ng)1nM3NEBNext High-Fidelity 2× PCR Master Mix2001x45BC-AG-f01 (100 μM)20.5uM55BC-AG-r01 (100 μM)20.5uM6Ultrapure distilled H2OUp to 400uL7Total volume400
?. [Library Amplification]
Split the premixture into 10 PCR tubes (40 μL per tube).
?. [Library Amplification]
Run the PCR reaction as follows:  ABCD1Cycle No.DenatureAnnealExtend2198oC, 2min32-13 (12 cycles)98oC, 15sec60oC, 20sec720C, 30sec41472oC, 5min
ABCD1Cycle No.DenatureAnnealExtend2198oC, 2min32-13 (12 cycles)98oC, 15sec60oC, 20sec720C, 30sec41472oC, 5min
?. [Library Amplification]
Combine the PCR products into a DNA LoBind tube.
?. [Library Amplification]
Add 200 μL of 6× gel loading dye (final conc. 2×) and mix the solution by vortexing.
?. [Library Amplification]
Run the sample on two 1.5% (wt/vol) TAE–agarose gels (30 mL of 5 × 6-cm mini gels with3-cm-width wells) and visualize the DNA using SYBR Safe DNA gel stain.
?. [Library Amplification]
Cut the DNA band (419 bp) using a blue-light Safe Imager.CRITICAL STEP We highly recommend using a blue-light Safe Imager, because the UVtransilluminator markedly decreases the recombination efficiency.
?. [Library Amplification]
Purify the DNA from the gel slice, using QIAquick Gel Extraction Kit according to themanufacturer’s protocol.
?. [Library Amplification]
Elute the DNA in 50 μL of Buffer EB per column. If multiple columns are used, combinethe eluate.
?. [Library Amplification]
Purify the DNA using 1.2 volumes of HighPrep PCR reagent and following Steps 6–17.
?. [Library Amplification]
Measure the DNA concentration using a NanoDrop spectrophotometer. The expected concentrationis ~25 ng/μL.PAUSE POINT Purified DNA can be stored at −20 °C for months.
?. [Library Amplification]
Dissolve the Agilent oligonucleotide (10 pmol) (Fig. 1b and Box 1) in 100 μL TE buffer to obtain a100 nM solution.
?. [Vector Linearization]
Set up the vector digestion reaction as follows:  ABC1ReagentVolume (uL)Final Conc.2pLS-SceIVariable (10ug)3CutSmart (10x)201x4AgeI-HF (20U/uL)50.5U/uL5SbfI-HF (20U/uL)50.5U/uL6Ultrapure distilled H2Omake up to 200uL7Total volume200
ABC1ReagentVolume (uL)Final Conc.2pLS-SceIVariable (10ug)3CutSmart (10x)201x4AgeI-HF (20U/uL)50.5U/uL5SbfI-HF (20U/uL)50.5U/uL6Ultrapure distilled H2Omake up to 200uL7Total volume200
?. [Vector Linearization]
Incubate the reaction at 37 °C for 3 h to overnight.
?. [Vector Linearization]
To complete the plasmid digestion, add 5 μL of AgeI-HF (20 U/μL) and 5 μL of SbfI-HF (20 U/μL)to the reaction.
?. [Vector Linearization]
33 Incubate the reaction at 37 °C for 3 h to overnight.
?. [Vector Linearization]
Vortex for 30 s and incubate at 80 °C for 20 min.
?. [Vector Linearization]
Purify the DNA using 0.65 volume (136.5 μL) of HighPrep PCR reagent and following Steps 6–17.
?. [Vector Linearization]
Measure the DNA concentration using a NanoDrop spectrophotometer.
?. [Vector Linearization]
To check the DNA size and quality, run 100–200 ng of the linearized vector and purified insertDNA (from Step 29) on a 1% (wt/vol) gel along with a 1-kb DNA ladder. Make sure that specificsingle bands (7.8-kb linearized vector and 419-bp insert DNA) appear, but that no other bandsappear on the gel.
?. [Recombination and electroporation]
Set up the recombination reaction as follows:  ABC1ReagentVolume (uL)Final Conc.2Linearized pLS-SceI (from step 36)Variable (1 μg)3Purified insert DNA (from step 29)Variable (250 ng)4NEBuilder HiFi DNA Assembly Master Mix1001X5Ultrapure distilled H2OMake up to 200 μL6Total volume200
ABC1ReagentVolume (uL)Final Conc.2Linearized pLS-SceI (from step 36)Variable (1 μg)3Purified insert DNA (from step 29)Variable (250 ng)4NEBuilder HiFi DNA Assembly Master Mix1001X5Ultrapure distilled H2OMake up to 200 μL6Total volume200
?. [Recombination and electroporation]
Incubate the reaction at 37 °C for 1 h.
?. [Recombination and electroporation]
Purify the DNA using 1.8 volume (90 μL) of HighPrep PCR reagent and following Steps 6–14.
?. [Recombination and electroporation]
Place the tube on the magnet for 1–2 min and transfer 18 μL of the eluate to a DNA LoBind tube.
?. [Recombination and electroporation]
Measure the DNA concentration using a NanoDrop spectrophotometer. Make sure the DNA the concentration of the recombination product (the eluate) is >25 ng/μL, so as not to need to add>4 μL to 100 μL of competent cells in Step 50.PAUSE POINT The recombinant product can be stored at −20 °C for at least a month.
?. [Recombination and electroporation]
Thaw NEB 10-beta electrocompetent cells on ice. We usually use 100 μL (one tube) of thecompetent cells for low-complexity libraries (0.5–2 million total barcodes) and 400 μL (four tubes)for high-complexity libraries (8–12 million total barcodes).CRITICAL STEP Competent cells and cuvettes should be kept on ice during the following procedure.
?. [Recombination and electroporation]
Prewarm 4–5 mL of Stable Outgrowth Medium (from the NEB 10-beta electrocompetent cells) at37 °C for at least 30 min.
?. [Recombination and electroporation]
Add 100 ng of the recombination product per 100 μL competent cells. The volume of DNA shouldbe
?. [Recombination and electroporation]
Gently transfer 50 μL of the competent cells to a 1-mm-gap cuvette without creating bubbles. Twocuvettes are prepared for 100 μL of competent cells.
?. [Recombination and electroporation]
Gently tap the cuvettes on the counter to move the cells to the bottom.
?. [Recombination and electroporation]
Place the cuvettes in a Gemini X2 electroporator and shock the cells with the following settings:voltage, 2.0 kV; resistance, 200 ohms; capacitance, 25 μF, number of pulses, 1; gap width, 1 mm.
?. [Recombination and electroporation]
Immediately add 450 μL of prewarmed Stable Outgrowth Medium to the cuvettes, thoroughly mixby pipetting up and down, and transfer to a 14-mL conical tube.
?. [Recombination and electroporation]
Repeat the electroporation for all cuvettes, combining the electroporated bacteria in a single tube(total 1 mL culture per 100 μL competent cells).
?. [Recombination and electroporation]
Add fresh Stable Outgrowth Medium and scale up to 4 mL in total. If 400 μL competent cells wereused, there is no need to add more.
?. [Recombination and electroporation]
Incubate the cells at 37 °C for 1 h with agitation (200 r.p.m.). Prewarm ten 15-cm LB agar plates at37 °C.CRITICAL STEP We recommend using 15-cm plates rather than larger plates because theseenable fine-tuning of the colony numbers collected.
?. [Recombination and electroporation]
Dilute 2 μL of the bacteria in 400 μL of fresh LB medium in a 1.5-mL tube and plate the entire tubeof diluted bacteria (402 μL) in a prewarmed 15-cm LB agar plate along with 20 μL of 100 mg/mLcarbenicillin. This plate will be used for colony counting and plasmid mini prep.
?. [Recombination and electroporation]
Plate undiluted bacteria onto the other nine prewarmed 15-cm LB agar plates (400 μL/plate), alongwith 100 μL/plate of 100 mg/mL carbenicillin. A higher amount of carbenicillin than usual is addedbecause the dense culture conditions increase the risk of non-transformed bacteria growth.
?. [Recombination and electroporation]
Incubate the plates at 37 °C overnight.
?. [Recombination and electroporation]
To check the plasmid sequence of individual colonies, pick 16 colonies from the diluted-bacteriaplate, purify the plasmids using a QIAprep Spin Miniprep Kit, and send them for Sangersequencing using n40.dn.F and EGFP.up.R primers (Supplementary Table 3). Confirm that thesequence structure corresponds to the design (Fig. 1e, Extended Data Fig. 1c).PAUSE POINT The plates can be stored at 4 °C for a month.? TROUBLESHOOTING
?. [Colony counting and plasmid library prep]
Count the number of colonies on the diluted-bacteria plate. If there are too many colonies, countcolonies in a quarter of the area and multiply by four to estimate the total number of colonies onthe plate.
?. [Colony counting and plasmid library prep]
Estimate the total number of colonies per undiluted-bacteria plate by multiplying the colony countin the diluted-bacteria plate by 200 (Supplementary Table 1).
?. [Colony counting and plasmid library prep]
Determine the number of undiluted-bacteria plates to be used for the following plasmid preps. Thetotal number of colonies needed can be determined by multiplying the number of designed CRSs bythe desired number of barcodes per CRSCRITICAL STEP The ideal number of barcodes per CRS is between 50 and 200. Fewer barcodesper CRS may reduce reproducibility. More barcodes per CRS requires more cells, more virus anddeeper sequencing reads, which increase costs. In addition, associating >200 barcodes per CRS doesnot increase reproducibility.
?. [Colony counting and plasmid library prep]
Add 5–6 mL of LB medium to each bacterial plate and gently scrape the colonies, using a cell lifterwithout disturbing solid agarose.
?. [Colony counting and plasmid library prep]
Collect the bacterial suspension and combine into a few 50-mL tubes.
?. [Colony counting and plasmid library prep]
Add 5–6 mL of fresh LB medium again to the plates and collect as much leftover bacteria aspossible into the tubes.
?. [Colony counting and plasmid library prep]
Purify the plasmids using a Qiagen Plasmid Plus Midi Kit, following the ‘standard protocol’in the manufacturer’s protocol. The number of columns to be used depends on the amount ofbacteria. We usually use four columns of Qiagen Plasmid Plus Midi Kit per undilutedbacteriaplate.
?. [Colony counting and plasmid library prep]
Measure the plasmid concentration using a NanoDrop spectrophotometer. The expectedconcentration is 0.5–2 μg/μL.
?. [Colony counting and plasmid library prep]
To check the DNA size and quality, run 100–200 ng of the plasmid on a 1% (wt/vol) agarose gelalong with a 1-kb DNA ladder.PAUSE POINT The purified plasmid library can be stored at −20 °C for years.
?. [Sequencing for CRS–barcode association]
Set up the PCR reaction. This reaction adds a P5 flowcell sequence and the sample index sequenceupstream and a P7 flowcell sequence downstream of the CRS–barcode fragment . NOTICE: Use different sample index sequences for pLSmP-ass-i# if multiple libraries aregenerated and multiplexed  ABC1ReagentVolume (uL)Final Conc.2Plasmid libraryVariable (40ng)3NEBNext High-Fidelity 2× PCR Master Mix1001x4pLSmP-ass-i# (100 μM)10.5uM5pLSmP-ass-gfp (100 μM)10.5uM6Ultrapure distilled H2OMake up to 200uL7Total volume200
ABC1ReagentVolume (uL)Final Conc.2Plasmid libraryVariable (40ng)3NEBNext High-Fidelity 2× PCR Master Mix1001x4pLSmP-ass-i# (100 μM)10.5uM5pLSmP-ass-gfp (100 μM)10.5uM6Ultrapure distilled H2OMake up to 200uL7Total volume200
?. [Sequencing for CRS–barcode association]
Split the premixture into five PCR tubes (40 μL per tube).
?. [Sequencing for CRS–barcode association]
Run the PCR reaction as follows:  ABCD1Cycle No.DenatureAnnealExtend2198oC, 1min32-16 (15 cycles)98oC, 15sec60oC, 20sec720C, 3min41772oC, 5minCRITICAL STEP Incomplete DNA elongation may create chimeric DNA annealing in the nextcycle and can cause CRS–barcode swapping. A longer extension time (3 min) can help to reducethis risk.
ABCD1Cycle No.DenatureAnnealExtend2198oC, 1min32-16 (15 cycles)98oC, 15sec60oC, 20sec720C, 3min41772oC, 5min
?. [Sequencing for CRS–barcode association]
Combine the PCR products in a DNA LoBind tube.
?. [Sequencing for CRS–barcode association]
Add 100 μL of 6× gel loading dye (final conc. 2×) and mix the solution by vortexing.
?. [Sequencing for CRS–barcode association]
Run the sample on a 1.5% (wt/vol) agarose gel (30 mL of 5 cm × 6-cm mini gel with 3-cm widthwell).
?. [Sequencing for CRS–barcode association]
Cut the DNA band (470 bp) using a blue-light Safe Imager.
?. [Sequencing for CRS–barcode association]
Purify the DNA from the gel slice using the QIAquick Gel Extraction Kit according to themanufacturer’s protocol.
?. [Sequencing for CRS–barcode association]
79 Elute the DNA in 50 μL Buffer EB per column. If multiple columns are used, combine the eluate.
?. [Sequencing for CRS–barcode association]
Purify the DNA using 1.8 volumes of HighPrep PCR reagent, following Steps 6–17.
?. [Sequencing for CRS–barcode association]
Measure the DNA concentration using Qubit dsDNA HS Assay Kit according to the manufacturer’sprotocol.
?. [Sequencing for CRS–barcode association]
To check the DNA size and quality, run 50–100 ng of the DNA on a 1.5% (wt/vol) agarose gel alongwith a 100-bp DNA ladder.PAUSE POINT Purified DNA can be stored at −20 °C for months.
?. [Sequencing for CRS–barcode association]
Send the purified DNA and custom primers for sequencing.The sequencing should be done using paired-end reads covering the full CRS with some overlap (here 146 bp each), with 15 cycles for index read 1 and 10 cycles for index read 2. Index read 1 provides the barcode sequence, and index read 2 provides the sample index Let the sequencing facility know that index read 2 should be used for demultiplexing and that short reads should not be masked, bcl2fastq parameters: --minimum-trimmed-read-length 0 --mask-short-adapter-reads 0. A minimum 10× coverage of sequencing reads based on the total number of barcodes is required. For example, we use an Illumina MiSeq v.2 run (15 million reads) for a 0.5-million-barcode library or an Illumina NextSeq mid-output run(120 million reads) for an 8- to 12-million-barcode library  ABCD1READCYCLESPRIMEROUTPUT2Read 1146pLSmP-ass-seq-R1CRS (upstream, forward)3Read 2146pLSmP-ass-seq-R2CRS (downstream, reverse)4Index read 115pLSmP-ass-seq-ind1Barcode (forward)5Index read 210pLSmP-rand-ind2Sample index
ABCD1READCYCLESPRIMEROUTPUT2Read 1146pLSmP-ass-seq-R1CRS (upstream, forward)3Read 2146pLSmP-ass-seq-R2CRS (downstream, reverse)4Index read 115pLSmP-ass-seq-ind1Barcode (forward)5Index read 210pLSmP-rand-ind2Sample index
?. [Lentivirus infection and DNA/RNA extraction]
Determine the number of integrations per barcode (i.e., total number of a particular barcodeexisting in an entire cell population)Use the spreadsheet provided in Supplementary Table 1. https://static-content.springer.com/esm/art%3A10.1038%2Fs41596-020-0333-5/MediaObjects/41596_2020_333_MOESM2_ESM.xlsxWe recommend a range between 50 and 500 integrations per barcode. Fewer numbers increase the riskof barcode loss during the downstream procedure. Higher numbers are better but increase the cost.
?. [Lentivirus infection and DNA/RNA extraction]
Seed an appropriate number of cells in 10-cm or 15-cm dishes. The number of cells required is determined as total barcode integrations (total number of any barcodes existing in the entire cell population) divided by the MOI of the cells (Supplementary Table 1). Three independent biological replicates should be performed, and each replicate sample should be treated separately during the following procedures
?. [Lentivirus infection and DNA/RNA extraction]
Incubate the cells overnight.
?. [Lentivirus infection and DNA/RNA extraction]
Refresh the culture media (culture conditions depend on the cell type used) and add Polybrene atthe appropriate concentration (Box 2).
?. [Lentivirus infection and DNA/RNA extraction]
111 Add an appropriate amount of the lentivirus library. The volume of virus required is given as totalbarcode integrations divided by virus titer (Supplementary Table 1).
?. [Lentivirus infection and DNA/RNA extraction]
Refresh the culture media (culture conditions depend on the cell type used) with no Polybrene thefollowing day.
?. [Lentivirus infection and DNA/RNA extraction]
After 2 d (3 d of culture in total), check GFP fluorescence to confirm proper lentiviral integrationand expression. GFP expression depends on library design. If the majority of the CRSsin the library are active enhancers, the cells should have strong GFP expression.
?. [Lentivirus infection and DNA/RNA extraction]
Remove the culture media and wash the cells with DPBS three times. Remove the DPBS completely.
?. [Lentivirus infection and DNA/RNA extraction]
Add RLT Plus lysis buffer (from the AllPrep DNA/RNA Mini Kit) supplemented with 2-mercaptoethanol (10 μL of 2-mercaptoethanol per 1 mL of RLT Plus). We usually use 1,200 μL or 2,400 μL of lysis buffer per 10-cm dish or 15-cm dish, respectively.
?. [Lentivirus infection and DNA/RNA extraction]
Scrape the cells using a cell lifter and homogenize the cell lysis, using a 3-mL syringe and 20-gaugeneedle.PAUSE POINT The cell lysate can be frozen and stored at −80 °C for months.
?. [Lentivirus infection and DNA/RNA extraction]
Transfer the lysate to DNA columns. Use two or four columns per 10-cm dish or 15-cm dish,respectively
?. [Lentivirus infection and DNA/RNA extraction]
Extract genomic DNA and total RNA simultaneously using the AllPrep DNA/RNA Mini Kit accordingto the manufacturer’s protocol. For RNA samples, perform DNase treatment between two of the 350-μLRW1 washes using Qiagen’s RNase-free DNase Set, according to the manufacturer’s protocol
?. [Lentivirus infection and DNA/RNA extraction]
Elute DNA in 30 μL/column of Buffer EB and combine the eluates of each replicate in a single tube.Keep each of the three replicates separate.
?. [Lentivirus infection and DNA/RNA extraction]
Elute RNA in 30 μL/column of RNase-free H2O and combine the eluates of each replicate in a singletube. Keep each of the three replicates separate
?. [Lentivirus infection and DNA/RNA extraction]
Measure the concentrations of the DNA and RNA samples using a NanoDrop spectrophotometer. Atleast 12 μg DNA and 60 μg RNA per replicate are required.PAUSE POINT DNA can be stored at −20 °C for years. RNA can be stored at −80 °C for months
?. [Reverse transcription]
Treat RNA samples with DNase, using the TURBO DNA-free Kit and following the manufacturer’sprotocol for ‘Rigorous DNase treatment’. Because the RNA sample has already been treated withDNase during the AllPrep procedure (Step 93), TURBO DNase treatment can be done using ahigh-concentration condition (without any dilution).
?. [Reverse transcription]
Measure the RNA concentration using the Qubit RNA HS Assay Kit. At least 60 μg or 240 μg totalRNA per replicate is required for a low- (0.5–2 million total barcodes) or high- (8–12 million totalbarcodes) complexity library, respectively.PAUSE POINT DNase-treated RNA can be stored at −80 °C for months
?. [Reverse transcription]
For RNA samples, perform a reverse-transcription reaction in 8-strip PCR tubes. This reaction addsa 16-bp UMI and a P7 flowcell sequence downstream of the barcode.For a low-complexity library (0.5–2 million total barcodes), use the amounts given in the table below. Fora high-complexity library (8–12 million total barcodes), we recommend multiplying all amounts inthe following table by 4  ABC1ReagentVolume (μL)Final conc.2RNAVariable (60 μg total RNA)3P7-pLSmP-ass16UMI-gfp (100 μM)0.250.25 μM4dNTP mix (10 mM, from SuperScript II Reverse Transcriptase)50.5 mM5UltraPure distilled H2OMake up to 65 μL6Total volume65uL
ABC1ReagentVolume (μL)Final conc.2RNAVariable (60 μg total RNA)3P7-pLSmP-ass16UMI-gfp (100 μM)0.250.25 μM4dNTP mix (10 mM, from SuperScript II Reverse Transcriptase)50.5 mM5UltraPure distilled H2OMake up to 65 μL6Total volume65uL
?. [Reverse transcription]
Incubate the reaction at 65 °C for 5 min using a thermal cycler.
?. [Reverse transcription]
Place the tubes on ice.
?. [Reverse transcription]
Add 20 μL 5× First Strand buffer (from SuperScript II Reverse Transcriptase) and 10 μL 0.1 M DTT(from SuperScript II Reverse Transcriptase).
?. [Reverse transcription]
Incubate the tubes at 42 °C for 2 min using a thermal cycler.
?. [Reverse transcription]
Add 5 μL of Superscript II.
?. [Reverse transcription]
130 Incubate the tubes at 42 °C for 50 min, followed by incubation at 70 °C for 15 min using a thermal cycler.PAUSE POINT cDNA can be stored at −20 °C for months.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Dilute DNA samples (from Step 121) to a final concentration of 120 ng/μL. For RNA samples(from Step 96; we refer to RT products as RNA samples in the downstream steps to distinguishthem from samples derived from genomic DNA), use all 100 μL of RT products for the followingfirst-round PCR reaction.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Perform a first-round PCR reaction with all three replicates of both DNA and RNA samples. This reaction adds the P5 flowcell sequence and sample index sequence upstream and a 16-bp UMI and P7flowcell sequence downstream of the barcode . Use different sample index sequences for each samplehttps://static-content.springer.com/esm/art%3A10.1038%2Fs41596-020-0333-5/MediaObjects/41596_2020_333_MOESM4_ESM.xlsxFor a low-complexity library (0.5–2 million total barcodes), use the amounts given in the table below. For a high-complexity library (8–12 million total barcodes), we recommend multiplying all amounts in the following table by 4.  ABC1ReagentVolume (μL)Final conc.2DNA or cDNA100 (12 μg DNA or entire RT product)3NEBNext High-Fidelity 2× PCR Master Mix2001x4P7-pLSmP-ass16UMI-gfp (100 μM)20.5 μM5P5-pLSmP-5bc-i# (100 μM)20.5 μM6UltraPure distilled H2O967Total volume400Split the premixture into 8 PCR tubes (50 μL per tube).  ABC1ReagentVolume (μL)Final conc.2DNA or cDNA400 (48 μg DNA or entire RT product)3NEBNext High-Fidelity 2× PCR Master Mix8001x4P7-pLSmP-ass16UMI-gfp (100 μM)80.5 μM5P5-pLSmP-5bc-i# (100 μM)80.5 μM6UltraPure distilled H2O3847Total volume1600Split the premixture into 32 PCR tubes (50 μL per tube). 4x 8-strips
ABC1ReagentVolume (μL)Final conc.2DNA or cDNA100 (12 μg DNA or entire RT product)3NEBNext High-Fidelity 2× PCR Master Mix2001x4P7-pLSmP-ass16UMI-gfp (100 μM)20.5 μM5P5-pLSmP-5bc-i# (100 μM)20.5 μM6UltraPure distilled H2O967Total volume400
ABC1ReagentVolume (μL)Final conc.2DNA or cDNA400 (48 μg DNA or entire RT product)3NEBNext High-Fidelity 2× PCR Master Mix8001x4P7-pLSmP-ass16UMI-gfp (100 μM)80.5 μM5P5-pLSmP-5bc-i# (100 μM)80.5 μM6UltraPure distilled H2O3847Total volume1600
?. [Library prep and sequencing for RNA and DNA barcode counts]
Run the PCR reaction as follows.  ABCD1CycleDenatureAnnealExtend2198oC, 1min32-4 (3 cycles)98oC, 10sec60oC, 30sec72oC, 1min4572oC, 5min
ABCD1CycleDenatureAnnealExtend2198oC, 1min32-4 (3 cycles)98oC, 10sec60oC, 30sec72oC, 1min4572oC, 5min
?. [Library prep and sequencing for RNA and DNA barcode counts]
After the PCR reaction, combine all eight tubes for each sample in a DNA LoBind tube.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Purify the DNA, using 1.8 volumes (700 μL) of HighPrep PCR reagent and following Steps 6–14.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Add 60 μL of Buffer EB to the beads and elute the DNA by pipetting and vortexing.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Place the tube on the magnet for 1–2 min and transfer 55–58 μL of the eluate to a LoBind tube.Store the tubes on ice.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Set up the preliminary PCR reaction for each sample in a 96-well qPCR plate. This run finds thenumber of PCR cycles required for the following second-round PCR reaction.  ABC1ReagentVolume (μL)Final conc.2First-round PCR product (from step 112)53NEBNext High-Fidelity 2× PCR Master Mix101x4P7 (100uM)0.10.5 μM5P5 (100uM)0.10.5 μM6SYBR green (100X)0.17UltraPure distilled H2O4.78Total volume20Run the qPCR reaction using a qPCR instrument as follows  ABCD1CycleDenatureAnnealExtend2198oC, 1min32-31 (30 cycles)98oC, 10sec60oC, 30sec72oC, 1min
ABC1ReagentVolume (μL)Final conc.2First-round PCR product (from step 112)53NEBNext High-Fidelity 2× PCR Master Mix101x4P7 (100uM)0.10.5 μM5P5 (100uM)0.10.5 μM6SYBR green (100X)0.17UltraPure distilled H2O4.78Total volume20
ABCD1CycleDenatureAnnealExtend2198oC, 1min32-31 (30 cycles)98oC, 10sec60oC, 30sec72oC, 1min
?. [Library prep and sequencing for RNA and DNA barcode counts]
On the basis of the raw amplification curve of each sample, determine the number of cycles atwhich the amplification nearly plateaus for each sample.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Set up the second-round PCR reaction for each sample as follows. Because it is expected that th numbers of cycles required for DNA and RNA samples will be different, we recommend running the PCRs for them separately. For a low-complexity library (0.5–2 million total barcodes), use theamounts given in the table below. For a high-complexity library (8–12 million total barcodes), werecommend multiplying all amounts in the following table by 4.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Set up the second-round PCR reaction for each sample as follows. Because it is expected that thenumbers of cycles required for DNA and RNA samples will be different, we recommend runningthe PCRs for them separately. For a low-complexity library (0.5–2 million total barcodes), use theamounts given in the table below. For a high-complexity library (8–12 million total barcodes), werecommend multiplying all amounts in the following table by 4.  ABC1ReagentVolume (μL)Final conc.2DNA or cDNA503NEBNext High-Fidelity 2× PCR Master Mix1001x4P7 (100 μM)10.5 μM5P5 (100 μM)10.5 μM6UltraPure distilled H2O487Total volume200Split the premixture into 5 PCR tubes (40 μL per tube).  ABC1ReagentVolume (μL)Final conc.2DNA or cDNA503NEBNext High-Fidelity 2× PCR Master Mix4001x4P7 (100 μM)40.5 μM5P5 (100 μM)40.5 μM6UltraPure distilled H2O1927Total volume800Split the premixture into 20 PCR tubes (40 μL per tube). 4x 8-strips
ABC1ReagentVolume (μL)Final conc.2DNA or cDNA503NEBNext High-Fidelity 2× PCR Master Mix1001x4P7 (100 μM)10.5 μM5P5 (100 μM)10.5 μM6UltraPure distilled H2O487Total volume200
ABC1ReagentVolume (μL)Final conc.2DNA or cDNA503NEBNext High-Fidelity 2× PCR Master Mix4001x4P7 (100 μM)40.5 μM5P5 (100 μM)40.5 μM6UltraPure distilled H2O1927Total volume800
?. [Library prep and sequencing for RNA and DNA barcode counts]
Run the qPCR reaction using a qPCR instrument as followsAccording to cycles numbers established in step 113-114  ABCD1CycleDenatureAnnealExtend2198oC, 1min32-31 (X cycles)98oC, 10sec60oC, 30sec72oC, 1min
ABCD1CycleDenatureAnnealExtend2198oC, 1min32-31 (X cycles)98oC, 10sec60oC, 30sec72oC, 1min
?. [Library prep and sequencing for RNA and DNA barcode counts]
Combine each sample into a DNA LoBind tube.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Purify the DNA using 1.8 volume (360 μL) of HighPrep PCR reagent following Steps 6–14.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Elute the DNA in 20 μL EB.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Measure the DNA concentration using a NanoDrop spectrophotometer.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Pool three replicates of DNA samples or RNA samples using an equal amount (1 μg) from eachreplicate, using Supplementary Table 4. Keep DNA and RNA samples separated until these arepooled at the later step (Step 160).PAUSE POINT DNA can be stored at −20 °C for months.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Add equal volume of 6x gel loading dye (final conc. 3×) and mix the solution by vortexing.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Run the pooled sample on a 1.8% (wt/vol) agarose gel (30 mL of 5 cm ×6 cm mini gels with 1.3 cm-width well
?. [Library prep and sequencing for RNA and DNA barcode counts]
Cut the DNA bands (162 bp) using a blue-light Safe Imager.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Purify the DNA from the gel slices using QIAquick Gel Extraction Kit according to themanufacturer’s protocol. Use one column for each of DNA or RNA samples.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Elute the DNA in 50 μL Buffer EB per column.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Purify the DNA using 1.8 volume (90 μL) of HighPrep PCR reagent
?. [Library prep and sequencing for RNA and DNA barcode counts]
Add 20 μL of Buffer EB to the beads and elute the DNA by pipetting and vortexing..
?. [Library prep and sequencing for RNA and DNA barcode counts]
Place the tube on the magnet for 1-2 min and transfer 18 μL of the eluate to a LoBind tube.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Measure the DNA concentration using a Qubit dsDNA HS Assay Kit according to manufacturer’sprotocol.
?. [Library prep and sequencing for RNA and DNA barcode counts]
To check the DNA size and quality, run 20-30 ng of the DNA on a 1.8% (wt/vol) gel along with100-bp DNA ladder.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Pool the DNA and RNA samples in a single LoBind tube with 1:3 ratio to obtain 100 μL mixture atthe final concentration of 10 nM (1 ng/μL), using Supplementary Table 4.PAUSE POINT The pooled DNA can be stored at −20 °C for months.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Send the sequencing library and custom primers for sequencing The sequencing should be done with paired-end 15 bp (barcode length), 16 cycles for index read 1 and 10 cycles for index read 2. Index read 1 provides the UMI sequence, and index read 2 provides the sample index.let the sequencing facility know that the index read 2 should be used for demultiplexing and that short reads should not be masked, bcl2fastq parameters: --minimum-trimmed-read- length 0 --mask-short-adapter-reads 0An average of 10×(DNA) and 30×(RNA) coverage of the library (based on number of barcodes) viasequencing reads is required. For example, we use an Illumina NextSeq high-output run (400Mreads over 3 replicates) for the 0.5M barcode library or three runs (1.2B reads over three replicates)for the 8–12 million barcode library (Supplementary Table 1). Sequencing cycles, primers, andexpected output for each read is shown below  ABCD1READCYCLESPRIMEROUTPUT2Read 115pLSmP-ass-seq-ind1Barcode (forward)3Read 215pLSmP-bc-seqBarcode (reverse)4Index read 116pLSmP-UMI-seqUMI5Index read 210pLSmP-5bc-seq-R2Sample index
ABCD1READCYCLESPRIMEROUTPUT2Read 115pLSmP-ass-seq-ind1Barcode (forward)3Read 215pLSmP-bc-seqBarcode (reverse)4Index read 116pLSmP-UMI-seqUMI5Index read 210pLSmP-5bc-seq-R2Sample index
?. [Library prep and sequencing for RNA and DNA barcode counts]
Bring the HighPrep PCR reagent to RT for at least 30 min before use. Shake thoroughly to fullyresuspend the magnetic beads. Add 1 volume (200 μL) of HighPrep PCR reagent and mixthoroughly by pipetting up and down 6–8 times
?. [Library prep and sequencing for RNA and DNA barcode counts]
Incubate the mixture for 5 min at RT
?. [Library prep and sequencing for RNA and DNA barcode counts]
Place the tube on the magnet for 2–3 min until the solution clears and beads pull to the side ofthe wall
?. [Library prep and sequencing for RNA and DNA barcode counts]
Carefully remove the supernatant by pipetting without disturbing the beads. A small amount(10–20 μL) of supernatant can be left in the tube.
?. [Library prep and sequencing for RNA and DNA barcode counts]
With the tube on the magnet, add 500 μL of 80% (vol/vol) ethanol and incubate for 30 s.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Remove the ethanol by pipetting while the tube is still on the magnet.
?. [Library prep and sequencing for RNA and DNA barcode counts]
Repeat the 80% (vol/vol) ethanol washing
?. [Library prep and sequencing for RNA and DNA barcode counts]
Flash-spin (200–1,000g, 22–25 °C, 3 s) the tube, immediately place it back on the magnet andremove the supernatant
?. [Library prep and sequencing for RNA and DNA barcode counts]
Dry the bead pellet for 2–3 min. Do not overdry the beads
?. [Recombination and electroporation]
Place the tube on ice
?. [Recombination and electroporation]
Purify the DNA using 0.65 volume (136.5 μL) of HighPrep PCR reagent
?. [Recombination and electroporation]
Set up the digestion reaction to get rid of undigested vectors as follows:  ABC1ReagentVolume (uL)Final Conc.2Recombination product443CutSmart buffer (10×)51X4I-SceI (20 U/μL)1 (20U)0.4 U/uL5Total volume50
ABC1ReagentVolume (uL)Final Conc.2Recombination product443CutSmart buffer (10×)51X4I-SceI (20 U/μL)1 (20U)0.4 U/uL5Total volume50
?. [Recombination and electroporation]
Incubate the reaction at 37 °C for 1 h.
?. [Recombination and electroporation]
To elute the DNA, add 20 μL of Buffer EB to the beads and mix by pipetting and vortexing
?. [Lentivirus packaging]
Culture 293T cells in DMEM (with 10% heat-inactivated FBS).
?. [Lentivirus packaging]
Seed 10–12 million 293T cells per T225 flask. The number of flasks depends on the library  complexity and the infectability of the cells. For example, we use one flask for the 0.5-millionbarcode  library or six T225 flasks for the 8- to 12-million-barcode library when carrying out  lentiMPRA in HepG2 cells.
?. [Lentivirus packaging]
Incubate the cells for 2 d.
?. [Lentivirus packaging]
Prepare premixtures A and B as follows:  ● Premix A: 800 μL/flask OPTI-MEM and 60 μL/flask EndoFectin.  ● Premix B: 800 μL/flask OPTI-MEM, 10 μg/flask plasmid library, 6.5 μg/flask psPAX2, and 3.5 μg/flask pMD2.G.
?. [Lentivirus packaging]
Add premix A to premix B by pipetting and mix the mixture by inverting the tube.
?. [Lentivirus packaging]
Incubate the tube at RT for 15 min.
?. [Lentivirus packaging]
Add 1.6 mL/flask of the mixture to 293T cells (from Step 86).
?. [Lentivirus packaging]
Incubate the cells for 8–14 h.  ! CAUTION During the following procedure (Steps 92–103), the liquid and plastic waste should be  discarded into 10% (vol/vol) bleach, because they are contaminated with lentivirus. Culture plates  and tubes for storage should be clearly labeled as lentiviral contaminants.
?. [Lentivirus packaging]
Replace the media with 30 mL/flask DMEM (with 5% heat-inactivated FBS) supplemented with  1× ViralBoost reagent (60 μL reagent per 30 mL medium).
?. [Lentivirus packaging]
Incubate the cells for 36–48 h.
?. [Lentivirus packaging]
Check GFP expression using a fluorescence microscope. The majority of cells are expected to  express strong GFP, because viral RNA, including the GFP gene, is transcribed via the 5ʹ long  terminal repeat (LTR).
?. [Lentivirus packaging]
Filter the supernatant through a 0.45-μm PES filter system. Use multiple filters (one filter for up to  three T225 flasks), so as not to get clogged.
?. [Lentivirus packaging]
Transfer the flow-through into multiple 50-mL tubes (30 mL per tube).
?. [Lentivirus packaging]
Add 1/3 volume (10 mL per tube) of Lenti-X concentrator reagent, close the lid tightly and mix  gently by inverting the tubes.
?. [Lentivirus packaging]
Seal the lid with Parafilm and place the tube in a refrigerator at 4 °C for at least 4 h. PAUSE POINT The tubes can be stored at 4 °C up to 1 week.
?. [Lentivirus packaging]
Centrifuge the tubes at 1,500g for 45 min at 4 °C.
?. [Lentivirus packaging]
Discard the supernatant into 10% (vol/vol) bleach by gentle decanting.
?. [Lentivirus packaging]
Discard the remainder of the supernatant into 10% bleach by pipetting without disturbing the pellet.
?. [Lentivirus packaging]
Gently resuspend the pellet in cold DPBS. We usually use 600 μL DPBS per T225 flask.
?. [Lentivirus packaging]
Store the lentivirus at 4 °C. CRITICAL STEP We do not recommend freezing the virus, especially in the case of high complexity libraries, because freeze–thaw cycles substantially decrease the viral titer. We did not see amarked loss of the viral titer when stored at 4 °C for up to 3 weeks. The following infection experiments should be done within 3 weeks. PAUSE POINT The virus can be stored at 4 °C up to 3 weeks
?. [Lentivirus titration]
Infect the lentivirus library (0, 1, 2, 4, 8, 16, 32, 64 μL) into the cells to be used, extract genomic DNA from the cells, and perform qPCR as described in Box 2 (steps 1–14).
?. [Lentivirus titration]
Plot the MOI for each condition and draw a linear approximation with the virus volume on the x axis and the MOI on the y axis (Supplementary Table 2).https://static-content.springer.com/esm/art%3A10.1038%2Fs41596-020-0333-5/MediaObjects/41596_2020_333_MOESM3_ESM.xlsx
?. [Lentivirus titration]
On the basis of its slope and the number of cells seeded, calculate the virus titer (in transducing units per microliter), using Supplementary Table 1.https://static-content.springer.com/esm/art%3A10.1038%2Fs41596-020-0333-5/MediaObjects/41596_2020_333_MOESM2_ESM.xlsx