GSM5212186: PROseq_Treated_14D_rep2; Homo sapiens; OTHER
PRO-seq experiments were performed as described previously (Mahat, D. B. et al. 2016). Nuclei were isolated by Dounce homogenizer with loose pestle. 1x10^7 nuclei were subjected to nuclear run-on (30oC, 3min) in the presence of 25 μM Biotin-11-ATP/GTP/CTP/UTP (PerkinElmer). Total RNA was extracted and hydrolyzed in 0.2 M NaOH (on ice, 10 min). PRO-seq libraries used biotinylated nascent RNAs were purified by Dynabeads M-280 streptavidin (Invitrogen). Following adaptor ligation as described in previously (Mahat, D. B. et al. 2016), cDNA synthesis, and PCR amplification 140–350 bp libraries were size-selected by Pippin HT with 2% gel cassette 20B (Sage Science).