GSM8015014
Cell permeabilization were performed according to the protocol generated by the Nascent Transcriptomics Core at Harvard Medical School, Boston, MA. (https://ntc.hms.harvard.edu/resources) In PRO-seq, a run-on reaction is carried out with biotin-NTPs (either all 4, or one with additional unlabeled NTPs to reduce cost) and sarkosyl, is carried out in isolated nuclei or permeabilized cells. Incorporation of the single biotin-NTP halts further elongation of nascent RNA strands by RNAPII. The RNA strands are extracted, fragmented, and purified through streptavidin pull-down. Next, 3' adapters are ligated directly to the purified sample before another streptavidin purification step. The 5' ends are repaired using TAP and PNK before ligating 5' adapters. The adapter-flanked RNA fragments are enriched through another streptavidin pull-down process before RT and PCR amplification. The resultant cDNA strands are sequenced from the 3' end.