Error-prone PCR was performed as follows: 0.05U/uL Mutazyme II (Agilent, 200550), 1x Mutazyme II buffer, 1x SYBR, 200uM dNTP each, 300nM forward primer, 300nM reverse primer, 100pg Csde1 5'UTR fragment; 95°C 2min initial denaturation, 10 cycles of 95°C 30s, 63°C 20s, 72°C 1min. 100pg input amount was determined by initially varying the input amounts to determine the amount at which the PCR is in exponential phase (50% of signal plateau). These parameters result in a mutation rate of approximately 1 per 100nt. The error-prone PCR amplicon has homology arms for Gibson assembly into pcDNA5/FRT (ThermoFisher Scientific, V601020) plasmid with EGFP. The final construct has a flanking primer region for cDNA amplification upstream of the mutagenized 5'UTR and EGFP open reading frame downstream. NEB 10-beta E. coli cells (NEB, C3020K) are transformed by electroporation and plated over a total of 2000cm2 area to give ~10000 colonies. The plate is scraped and the mutagenesis library plasmid pool is purified. mESCs are grown, dissociated, and resuspended at 5x105 cells/mL. 3ug mutagenesis library plasmid pool DNA and 7.5uL Lipofectamine 2000 in 100uL OptiMEM (ThermoFisher Scientific, 31985062) are mixed with 2mL cells (1x106 cells) in mESC media. The cells are incubated for 10min in suspension, centrifuged, washed with mESC media, and plated into one well in a 6-well plate. 24 hours after transfection, the cells are washed, trypsinized and resuspended in 3.5mL mESC media. 1mL 1M bicine (Millipore Sigma, B3876), titrated to pH 8.5 at 25°C, is added to resuspended cells (250mM final bicine concentration). 500uL 16% dimethyl sulfate (MilliporeSigma, D186309) in ethanol is added (1.6% final concentration). Cells are mixed and incubated for 6min at 37°C. 2.5mL ice-cold 30% BME (MilliporeSigma, M3148) in ethanol is added to quench the reaction. Following centrifuge to remove the supernatant, the cells are lysed in Trizol (ThermoFisher Scientific, 15596026). Total RNA is phase extracted with chloroform and aqueous phase is purified on silica columns (Zymo, R1013). 10-20ug RNA is DNase treated at 37°C for 30min using 0.2U/uL TURBO DNase (ThermoFisher Scientific, AM2238) with 1U/uL Superase-In (ThermoFisher Scientific, AM2696) in 60uL and purified again on silica column. 1ug RNA is mixed with 1uL 1uM RT primer targeting the downstream EGFP and denatured in 6.25uL total volume (with H2O) at 65°C for 2min, then chilled to 4°C. Reverse transcription reaction conditions are as follows: 20mM Tris-HCl pH7.5, 75mM KCl, 10mM MgCl2, 5mM DTT, 500nM TGIRT (InGex, TGIRT50), 1U/uL Superase-In, 19uL total reaction volume. The RT reaction is preincubated for 25°C for 30min, then initiated with addition of 1uL 12.5mM dNTP each. After incubation at 60°C for 1hour, 1uL 2.5M NaOH is added and the reaction is heated at 95°C for 3min. 1uL 2.5M HCl and 1uL 500mM Tris-HCl pH 7.5 is added to neutralize. 29uL SPRIselect beads (Beckman Coulter, B23318) is used for purification of cDNA; elution volume is 7uL. For each of the 3 tiling primer pairs, PCR reaction is performed as follows: 0.2mM dNTP, 300nM forward primer, 300nM reverse primer, 1x SYBR Green I (ThermoFisher Scientific, S7563), Q5 Hot Start DNA polymerase (NEB, M0493S), 1x Q5 Hot Start Reaction Buffer, 1x Q5 Hot Start High GC Enhancer, 2uL cDNA, in total 20uL reaction volume. Cycling conditions are: 98°C 30s initial denaturation, 20 cycles of 98°C 10s, 64°C 10s, 72°C 30s. The amplicons are purified on silica columns and pooled. The pooled DNA is end-prepared, Illumina adaptor sequences are ligated, adaptor-ligated DNA is size selected with SPRIselect beads for 370bp, and 3 cycle barcoding PCR is performed (NEB, E7645S). 2x150bp paired end sequencing data is generated on Illumina HiSeq 4000 at Novogene.
GEO Accession
GSM4709647