GSM2773146: HUVEC-GROseq-oxPAPC; Homo sapiens; OTHER
Nuclei were extracted from 5-10 million cells and nuclear run-on reactions were performed for 5 minutes at 30°C in the presence of Sarkosyl and BrUTP. RNA was extracted with Trizol-LS. The RNA was base-hydrolyzed, dephosphorylated with PNK and purified using RNA Clean & Concentrator kit (Zymo). Poly(A)-tailing was followed by cDNA synthesis using complementary poly(T)-primers containing Illumina adapter sequences. Excess oligo was removed by Exonuclease I and cDNA fragments of were purified using ChIP DNA Clean & Concentrator kit. The recovered cDNA was RNaseH treated and circularized (CircLigase) and amplified for 13 cycles. The final product was ran on 10% TBE gel, gel purified (200-350 bp) and cleaned-up using ChIP DNA clean & Concentrator Kit.