title: GSM3030982: PRO-seq WT rep2 [YZ-2]; Mus musculus; OTHER
library_construction_protocol: Nuclei isolation by douncing in isotonic buffer. Nuclear run-on assays were performed with ATP, UTP, GTP, and biotin-11-CTP or with all 4 biotin-11-NTPs. Nuclei were lyzed with TRIzol, nuclear RNA was extracted and fragmented, and biotinylated RNA was purified with strepavidin magnetic beads, which was followed by 3' adaptor ligation, 5' repair, 5' adaptor ligation, reverse transcription, and PCR full-scale amplification. Briefly, 20 million nuclei were used for each run-on assay. In vitro nuclear run-on assays were carried out in the presence of 375 uM biotinylated NTPs and 0.5% Sarkosyl at 30°C for 3 min on the day of nuclei isolation. Total nuclear RNA was isolated by Trizol extraction and ethanol precipitation, and RNA pellets were kept in 75% ethanol at -80°C. On the next day, RNA was resuspended and hydrolyzed with 0.2 N NaOH, and biotinylated RNA was purified by streptavidin bead binding. Following adaptor ligation, RNA was reverse transcribed and PCR amplified. DNA libraries were PAGE purified and submitted for sequencing.