GSM8242423: VCaP_GRO_DHT_rep2; Homo sapiens; OTHER
Nuclei were extracted from 12.5 million cells and nuclear run-on reactions were performed for 5 minutes at 30°C in the presence of Sarkosyl and Br-UTP. RNA was extracted with Trizol, DNase-treated, base-hydrolyzed and dephosphorylated with PNK. Br-UTP-labeled run-on RNA was immunopurified with anti-BrdU-coated Protein G Dynabeads and precipitated overnight. Poly(A)-tailing was followed by cDNA synthesis using complementary poly(T)-primers. Excess oligo was removed by Exonuclease I and cDNA fragments of were purified with DNA purification kit. The recovered cDNA was circularized, linearized, amplified with 14 cycles. The final product was ran on 10% TBE gel, gel purified and cleaned-up with DNA purification kit.