title: GSM6369154: PD26_PROCAP_A; Homo sapiens; OTHER
library_construction_protocol: PRO-seq/CAP was performed on proliferating and senescent IMR90 cells as described in Mahat, D.B. et al. Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nat Protoc 11, 1455-76 (2016). Briefly, ~12e6 trypsinized cells were permeabilized and stored in storage buffer (10mM Tris-HCl pH 8.0, 25% glycerol, 5mM MgCl2, 0.1mM EDTA, 5mM DTT) at -80°C until use. The permeabilized cells were then resuspended in 1X nuclear run-on reaction mix containing biotin-11-rCTP and unlabeled rATP, rGTP and rUTP for 3 minutes at 37°C. RNA was extracted, fragmented and subjected to biotin enrichment. The enriched nascent RNA population was ligated to a 3' adapter sequence and subjected to a second round of biotin enrichment. The 5' ends of the RNA were decapped, phosphorylated, ligated to 5' adapter and subjected to a third biotin enrichment. The RNA was then reverse transcribed and PCR amplified with appropriate index primers. The libraries were purified by extraction from an 8% polyacrylamide gel, quantified by Bioanalyzer (Agilent) and qPCR (Kapa Biosystems). The PRO-seq/CAP run was performed on the NextSeq 500 platform (Illumina).