title: GSM1622613: K562-GROseq-C-rep2; Homo sapiens; OTHER
library_construction_protocol: 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, 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 11-14 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 HiSeq 2000.
attributes.tag: GEO Accession
attributes.value: GSM1622613