GRO-seq of HCT116 cells, Random-primed library prep DMSO 1 hr replicate 2
Run-on reactions were performed as in (Core et al, 2008). In brief, ice-cold isolated nuclei (100l) were added to 37C 100 L 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 30C, 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 based on the NEBNext Ultra II Directional Library Preparation Kit. 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 3 times. Samples were reverse-transcribed using random hexamers, and sequencing adapters added by PCR. 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