title: GSM3704351: Vehicle_1; Homo sapiens; RNA-Seq
library_construction_protocol: Cell culture media was removed and the monolayer was washed with DPBS.  Total RNA was extracted using a miRNeasy Mini Kit (Qiagen) using an on-column DNase I treatment, following the manufacturer's protocol. A Qubit 2.0 Fluorometer (Invitrogen) and an Agilent Technologies 2100 Bioanalyzer with an RNA nano chip were used to quantify and assess the quality of RNA, respectively.  Only samples with RNA Integrity Number > 8.0 were used.  NEBNext rRNA depletion modules (Catalog #: E6310L, New England Biosciences) were used to deplete rRNA on 500 ng of total RNA, according to manufacturer's instructions.  A NEBNext Ultra II Directional RNA kit (Catalog #: E7760, New England Biosciences) was used for library preparation according to the manufacturer's instructions.  RNA samples were then chemically fragmented, primed with random hexamers, and reverse transcribed to convert fragmented RNA to first strand cDNA.  The RNA template was removed and dUTP was incorporated in place of dTTP, after which the second strand of cDNA was synthesized by end repair and 3' end adenylation.  A hairpin loop adaptor was used to ligate an adaptor A corresponding T nucleotide on the hairpin loop adaptor was used to ligate an adaptor to the double-stranded cDNA.  Uracil-specific excision reagent (USER) enzyme was then used to remove the dUTP in the loop, as well as other incorporated U's in the second strand.  Final libraries were generated by PCR amplifying adaptor ligated DNA with Illumina barcoding primers, where fragments with both 5' and 3' adaptors would be enriched in the final PCR step.  Libraries were normalized to 2 nM, validated by a Bioanalyzer DNA chip, pooled equally, and then sequenced on a NextSeq500 v2.5 flow cell (1.8 pM) using paired-end chemistry (2 × 40 bp).  Approximately 20-25 million reads were generated per sample with a base quality score >Q30 (less than 1 error per 1000 bp).