GSM5509503: PRO-Seq_Pol_II_NTD_IAA_rep2; Mus musculus; OTHER
1-Biotin run-on was applied according to the protocol of Dig et al. (Dig et al., 2016). Total RNA was extracted using TRIzol reagent and fragmented by base hydrolysis. The nascent RNA labeled by Biotin were enriched with streptavidin beads and extracted using TRIzol reagent again. The enriched RNA was reverse transcribed into first-strand DNA using SuperScript IV reverse transcriptase kit (Invitrogen, 18090010) with N9 random primer. And RNA was eliminated by adding 2μl 1M NaOH and heating at 98℃ for 20min. DNA was purified with Phenol/Chloroform and precipitated with Ethanol. Finally, the DNA samples were subjected to TELP library construction (Peng et al., 2015). 200bp-400bp DNA libraries were gel-selected, purified, and subjected to HiSeq X-ten PE150 sequencing.