GRO-seq of HCT116 cells, Ligation-based library prep 10 μM Nutlin-3a 1hr replicate 2
Run-on reactions were performed as in (Core et al, 2008). In brief, ice-cold isolated nuclei (100l) were added to 30 C 100 uL reaction buffer (10mM Tris-Cl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCL, 20 units of SUPERase In, 1% sarkosyl, 500M ATP, GTP, and Br-UTP, 2M CTP). The reaction was allowed to proceed for 5 min at 30 C, followed by the addition of 23l of 10X DNAseI buffer, and 10l RNase free DNase I (Promega). 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 20l of DEPC-treated water. Libraries were prepared as in (Core et al, 2008). In brief, nascent RNA was extracted and fragmented by base hydrolysis in 0.2 N NaOH on ice for 1012 min, and neutralized by adding a 1 volume of 1 M Tris-HCl pH 6.8. Fragmented nascent RNA was purified using Anti-BrdU beads and ligated with reverse 3 RNA adaptor (/5Phos/GAUCGUCGGACUGUAGAACUCUGAAC/3InvdT/), and BrdU-labeled products were enriched by a second round of Anti-BrdU 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 Anti-BrdU 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.