GSM5384942: run-on-RNA #2; Mus musculus; OTHER
For nascent RNA, library-containing 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. First, nascent RNA still bound to magnetic beads was reverse transcribed with Superscript IV (Thermo Fisher), using a gene-specific primer in mKate2 that annealed 221nt downstream of the variable regions. cDNA from nascent RNA was removed from the beads with heat and then concentrated using 2x RNA XP beads (Beckman Coulter) to limit the number of parallel PCR reactions that had to be set up. Sequencing libraries were generated through 19 PCR cycles using NEBNext Ultra II Q5 (New England Biolabs) and NEBNext Multiplex oligos, uniquely indexing each sample. Amplicons were cleaned up using 1.5x AMPure XP beads (Beckman Coulter) and concentrations were quantified using the NEBNext Library Quant Kit for Illumina, before mixing indexed samples and checking the size distribution by TapeStation (Agilent).