GSM1973392: HCEC GROseq, rep2; Homo sapiens; OTHER
Cells of 1 to 3 million at 50-70% confluency were lysed by loading 1 ml of Tripure Isolation Reagent (Roche Diagnostics, 1667157). Two biological replica were prepared separately in each cell line. Total RNA was extracted by following manufacturer’s instruction. The extracted RNA was subjected on Bioanalyzer 2100 (Agilent Technologies) and RNA integrity number was confirmed as > 8. 10 ug each of total RNA was proceeded for capturing nascent transcript using Click-iT Nascent RNA capture kit (Life Technology, C10365) following manufacturer’s instruction. Briefly, rRNA was removed by Ribominus Eukaryote system v2 (Life Technology, A15020). Etynyl Uridine was crosslinked with azide modified biotin (PEG4 carboxiamide-6-azidohexanyl biotin) and recovered by streptavidin-coated magnetic beads. cDNA was prepared from EU labeled RNA by SuperScript VILO cDNA Synthesis Kit (Life Technology, 11754050). The sequence library was prepared using Illumina TruSeq Stranded mRNA Library Prep Kit (Illumina, RS-122-2101). The quality and quantity of the library were assessed on Bioanalyzer 2100 and Qubit (Life Technologies).