GSM4073590: parental_C; Homo sapiens; RNA-Seq
Total RNA was isolated with the use of TRI Reagent (T9424, Sigma) according to the manufacturer's instructions. RNA was subjected to TURBO DNase (AM2238, Ambion) treatment and depletion of nuclear-encoded rRNAs (RiboZero Kit, Epicenter). Libraries were constructed as previously described in Sultan et al., 2012 (PMID: 22609201) with minor modifications. Fragmentation and first strand cDNA synthesis were performed as in TruSeq RNA Library Prep kit v2 protocol (Illumina, RS-122-2001, instruction number 15026495 Rev. D), using SuperScript III reverse transcriptase (18080-085, Thermo Fisher Scientific). CutSmart buffer (NEB) was used for digestion with the USER Enzyme (NEB).  Enriched library quality was verified using 2100 Bioanalyzer and High Sensitivity DNA kit (5067-4626, Agilent). The libraries' concentration was estimated by qPCR means with KAPA Universal Library Quantification Kit (KK4824, Kapa Biosystems), according to the supplied protocol. Sequencing was carried out on an Illumina NextSeq 500 sequencing platform, using NextSeq 500 High Output Kit (150 cycles) (FC-404-1002, Illumina) and pair-end sequencing procedures of 2x75 cycles (Instructions: 15048776 Rev. D, 15046563 Rev. F).