GSM8070374: 81-008POST; Homo sapiens; RNA-Seq
For RNA extraction, snap-frozen tumor pieces (~50 mm3) were homogenized in TRizol reagent solution (Invitrogen, #AM9738) according to manufacturer's instructions using Precellys 24 Tissue Homogeneizer (Bertin technologies). Briefly, after homogenization, chloroform was added and the resulting mixture was centrifuged at 12,000g for 10 min at 4ºC. Aqueous phase was transferred to a clean tube and 70% ethanol was added. Then, total volume was filtered through RNeasy column (Qiagen, #74104) and RNA extraction was performed following protocol instructions. RNA quality was determined by Agilent's 2100 Bioanalyzer Lab-Chip technology. Total RNA samples (approximately 400 ng) were converted into cDNA sequencing libraries with the "QuantSeq 3'mRNA-Seq V2 Library Prep Kit (FWD) for Illumina" (Lexogen, #191). Briefly, library generation is initiated by reverse transcription with oligodT priming, followed by a random-primed second strand synthesis. An UMI (Unique Molecular Identifier) Second Strand Synthesis module was used, in which random primers featuring a 6 nt long Unique Molecular Identifier (UMI) tag are used (to be addressed for bias correction at data analysis). Primers from both steps contain Illumina-adapter sequences. Libraries were completed by PCR and sequenced on an Illumina NextSeq 550 instrument (with v2.5 reagent kits) by following manufacturer's protocols.