GSM8658240: W_1.2_rep2; Homo sapiens; RNA-Seq
Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer's protocol. RNA concentration and quality were assessed using a NanoDrop spectrophotometer and RNA integrity was determined using an Agilent Bioanalyzer or 4200 Tapestation. Samples with a RIN score >8.0 were used for library preparation. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was fragmented in reverse transcriptase buffer by heating to 94°C for 8 minutes. cDNA was synthesized using SuperScript III reverse transcriptase (Life Technologies) and random hexamers. A second strand synthesis reaction was performed to create double-stranded cDNA. Fragments were blunt-ended, an A base was added to the 3' ends, and Illumina sequencing adapters were ligated. The ligated fragments were amplified with primers containing unique dual index tags for 12-15 PCR cycles.