title: GSM5820993: WT mESC PRO-seq Rep 4; Mus musculus; OTHER
library_construction_protocol: mESCs were washed with PBS, brought into a single cell suspension with accutase, permeabilized and flash frozen. 1 million mESCs were subject to a nuclear run-on reaction containing only biotin-NTPs for 5 mins at 37C. Following the nuclear run-on, RNA was extracted by the Norgen Biotek Corp Total RNA Purification Kit (following manufacturer instructions) and fragmented by base hydrolysis. Nascent RNAs were enriched using prepared Dynabeads™ MyOne™ Streptavidin C1 (ThermoFisher) and trizol extracted. Following extraction, Isolated nascent RNAs underwent 3'end adapter ligation, biotin enrichment, 5'end decapping, 5'hydroxyl repair, on-bead reverse transcription, bead elution, and PCR amplifcation to 9-11 cycles