GSM5210291: MDA-MD-231 cells, WT, Thapsigargin, F10 (GROseq); Homo sapiens; OTHER
Cells were pre-treated with 5-ethynyl uridine (EU) for 30 min prior to nuclei fractionation using Sigma kit (#NUC101) followed by Invitrogen Click-iT™ Nascent RNA Capture Kit protocol for nascent transcript extraction (#C10365). RNA was then extracted using the RNeasy kit (Qiagen #74104) performing on-column DNA digestion for 15min and was subsequently reverse transcribed with Superscript VILO IV Master Mix from Invitrogen. Libraries were prepared following the protocol from NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (NEB #E7770S) and indexes (NEB #E7335S and #E7500S). The size of the libraries was confirmed using 4200 TapeStation and High Sensitivity D1K screen tape (Agilent Technologies) and their concentration was determined by qPCR based method using Library quantification kit (KAPA). The libraries were multiplexed and then sequenced on Illumina HiSeq2500 (Illumina) to generate 30M of single end 50 base pair reads.