PRO-seq of HCT116 cells, Ligation-based library prep DMSO 1 hr replicate 2
Run-on reactions were adapted from (Mahat et al, 2016). In brief, ice-cold isolated nuclei (100l) were added to 37C 100 L reaction buffer (Final Concentration: 5mM Tris-Cl pH 8.0, 2.5mM MgCl2, 0.5mM DTT, 150mM KCL, 10 units of SUPERase In, 0.5% sarkosyl, 125 M rATP, 125 M rGTP, 125 M rUTP, 25 M biotin-11-CTP. The reaction was allowed to proceed for 5 min at 37C. RNA was extracted twice with Trizol, washed once with chloroform, and precipitated with 3 volumes of ice-cold ethanol and 1-2 L GlycoBlue. The pellet was washed in 75% ethanol before resuspending in 20 l of DEPC-treated water. Nascent RNA was extracted and fragmented by base hydrolysis in 0.2 N NaOH on ice for 10-12 min, and neutralized by adding a 1 volume of 1 M Tris-HCl pH 6.8. Fragmented nascent RNA was purified using streptavidin beads and ligated with reverse 3 RNA adaptor (/5Phos/GAUCGUCGGACUGUAGAACUCUGAAC/3InvdT/), and biotin-labeled products were enriched by a second round of streptavidin bead binding and extraction. For 5' end repair, the RNA products were treated with tobacco acid pyrophosphatase (Epicenter) and T4 polynucleotide kinase (NEB). 5' repaired RNA was ligated to reverse 5' RNA adaptor (5' UGGAAUUCUCGGGUGCCAAGG) before being purified by a final round of streptavidin bead binding and extraction. RNA was reverse transcribed using 25 pmol of RP1 primer (5' AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA). The product was amplified 15 +/- 3 cycles and products >150 bp (insert > 70 bp) and size selected with 1X AMPure XP beads (Beckman) before being sequenced.