PRO-seq for Pol II occupancy project

Precision Run-On Sequencing (PROseq) performed as previously described (Kwak et al., 2013; Judd et al., 2020) with slight modifications. Data is obtained from 1) DNA methyltransferase triple knockout mouse embryonic stem cells (DNMT TKO mESCs) and 2) Drosophila Schneider's S2 cell line. For the Drosophila S2 cell samples, 10 million Drosophila S2 cells were used per sample with a spike-in of 0.1 million (1%) TKO mESCs. For the TKO mESC samples, 5 million TKO mESCs were used per sample with a spike-in of 0.05 million (1%) Drosophila S2 cells. Treatment samples were treated with 10 microM triptolide for various time points (2.5, 5, 10, or 20 minutes). Control samples were treated with DMSO for 20 minutes. PROseq data was obtained by permeabilising the cells, performing a 2 biotin run-on reaction using biotin-11-UTP and biotin-11-CTP and subsequent enrichments steps for biotinylated RNA during the library preparation