PRO-seq of MCF10A cells (WT p53 insertion at p53 locus), Template-Switching Reverse Transcription based library prep DMSO 3 hr replicate 1
Template-Switch Reverse Transcription protocol (also known as uPRO), was adapted from (Kim et al, 2019). Nuclei were incubated in the nuclear run-on reaction condition (5 mM Tris-HCl pH 8.0, 2.5 mM MgCl2, 0.5 mM DTT, 150 mM KCl, 0.5% Sarkosyl, 0.4 units / l of SUPERase-In) along with biotin-NTPs and rNTPs (125 M rATP, 125 M rGTP, 25 M rCTP, 125 M rUTP, and 25 M biotin-11-CTP) for 5 min at 37C. Run-On RNA was extracted using TRIzol, and fragmented with 0.2 N NaOH for 10-12 min on ice. Fragmented RNA was neutralized with 1 M Tris-HCl pH 6.8 , and buffer exchanged by passing through P-30 columns (Biorad). VRA3' RNA adaptor (/5Phos/GAUCGUCGGACUGUAGAACUCUGAAC/3InvdT/) is ligated at 5 M concentration for 1 hour at room temperature using T4 RNA ligase (NEB), and nascent RNA was enriched twice with streptavidin beads. Extracted RNA was converted to cDNA using template switch reverse transcription with 1 M RP1-short RT primer (5' GTTCAGAGTTCTACAGTCCGA), 3.75 M RTP-Template Switch Oligo (5' GCCTTGGCACCCGAGAATTCCArGrGrG), 1x Template Switch Enzyme and Buffer (NEB) at 42C for 30 min. Resulting product was size selected with AMPure XP beads, and the cDNA was PCR amplified using primers compatible with Illumina Small RNA sequencing (TruSeq Small RNA primers RP1 and RPIn).