title: GSM7310864: WT oleic acid rep3; Homo sapiens; RNA-Seq
library_construction_protocol: Following incubation with 200 µM OA (or vehicle control) overnight for 12 hr, total RNA was purified from cells using the RNeasy Mini Kit (#NC9677589, Qiagen). High-Capacity cDNA Reverse Transcription Kit (#4368814, Thermo Fisher) with random priming was used to obtain full-length reverse transcripts. Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation.  Library preparation was performed with 5 to 10ug of total RNA with a Bioanalyzer RIN score greater than 8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was then fragmented in reverse transcriptase buffer and heating to 94 degrees for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.