GSM8137050: OVCAR-8-STL001 rep1; Homo sapiens; RNA-Seq
Total RNA from cultured cells was extracted and purified using TRIzol reagent (Fisher Scientific) and the PureLink™ RNA Mini Kit (Fisher Scientific) including on-column DNase (Thermo Fisher Scientific) treatment according to the manufacturer's instructions. To assess the integrity of RNA, all samples were analyzed on the Agilent 4200 TapeStation (Agilent Technologies, USA). The remaining DNA concentrations were measured using the Qubit fluorometer (Thermo Fisher Scientific). In all the samples the DNA amounts did not exceed 2% of the total amount of nucleic acid. Sequencing libraries for Illumina sequencing platform were created in one batch in a 96-well plate, we used Stranded CORALL Total RNA-Seq Library Prep kit (Lexogen, Austria) with Lexogen's RiboCop HMR rRNA Depletion Kit. In brief, in the first step during rRNA removal we used 260–660 ngs of total RNA, and then followed by library creation initiated with random oligonucleotide primer hybridization with complementary sequence within the RNA template and reverse transcription. No prior RNA fragmentation was done before reverse transcription, as the insert size was determined by proprietary size-restricting method. Next, Illumina-compatible P5 sequences and UMIs (Unique Molecular Identifiers) were ligated at the 3′ end of the first-strand cDNA fragments. During the following steps of the 2nd-strand cDNA synthesis and the double-stranded cDNA amplification, unique i5 and i7 index sequences as well as complete adapter sequences required for cluster generation were added. The number of PCR amplification cycles was 12, as determined by qPCR using a small pre-amplification library aliquot for each sample.