GSM1232932: ThioMac-WT-GroSeq-KLA-12h; Mus musculus; OTHER
Nuclei were extracted from 8-12 million cells grown on 10 cm plates 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, DNase-treated, base-hydrolyzed and dephosphorylated with PNK. BrUTP-labeled run-on RNA was immunopurified with anti-BrdUTP-coated agarose beads 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 on a denaturing 10% polyacrylamide TBE-urea gel. The recovered cDNA was circularized, linearized, amplified for 10-16 cycles. The final product was ran on 10% TBE gel, gel purified and cleaned-up using ChIP DNA clean & Concentrator Kit. The final libraries were sequenced using an Illumina Genome Analyzer II or HiSeq 2000.
GEO Accession
GSM1232932