GSM5949848: mESC_BRM014_4h_PRO-seq_rep2; Mus musculus; OTHER
For nascent RNA, cells were permeabilized and run on mostly as described (Reimer et al, 2021), but with a few changes. For permeabilization, buffers W and P were supplemented with 1 mM EGTA, and buffer F was supplemented with 1 mM EDTA. Cells were resuspended in 500 µL Buffer W, 10 mL Buffer P was added, and this was incubated on a Nutator for 1 minutes, then 6 minutes on ice. Cells were washed once with 10mL buffer W, before being resuspended in Buffer F. Run-on was performed on 30M permeabilized cells, and in the 2X run-on reaction mix MgCl2 was present at 10 mM, SUPERase-In at 0.4 U/µL and (biotin-)rNTPs are at 50 µM each. Run-on was performed at 37°C for 15 minutes, and Biotin-11-C/UTP (Perkin-Elmer) were mixed with regular rGTP and rATP (New England BioLabs). After RNA isolation using the Total RNA Purification Kit (Norgen Biotek Corp), biotinylated RNAs were immediately bound to Streptavidin M-280 magnetic beads (ThermoFisher) and binding and washing was done as described.