title: GSM2828780: PRO-seq from Senescent Fibroblasts; Homo sapiens; OTHER
library_construction_protocol: PRO-seq was performed as described previously (Mahat et al., Nature Methods 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 then 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 run was performed on the NextSeq 500 platform (Illumina).
identifiers.namespace: GEO
identifiers.id: GSM2828780