GSM9594949: Whole Blood, ID13, D2, Placebo, 6hour; Homo sapiens; RNA-Seq
Whole blood RNA was isolated from PAXgene tubes for 221 study samples using the QIAsymphony PAXgene Blood RNA Kit and the QIAsymphony SP instrument (both Qiagen) according to the manufacturer instructions including DNase I digestion and Proteinase K treatment.  RNA concentration was determined by Quant-iT RiboGreen assay (ThermoFisher) using a Biotek Synergy H1 plate reader (Agilent Technologies) and RINs assessed using Agilent 4200 TapeStation (Agilent). RNA-seq libraries were prepared from 500 ng of total RNA using Universal Plus mRNA-seq Kit with NuQuant Human Globin AnyDeplete kit (Tecan Genomics) on a Biomek Fx robot (Beckman). First, messenger RNA transcripts were captured, fragmented, and converted to complementary DNA. Following second-strand synthesis and end repair, DNA fragments were ligated with dual-indexed adapters compatible with the Illumina platform. Libraries were subjected to strand selection and removal of globin transcripts, followed by 14 cycles of amplification. Purified libraries were analyzed with the Biotek Synergy H1 plate reader and Agilent 4200 TapeStation (Agilent Technologies) to assess concentration and size distribution, respectively, then normalized and pooled for sequencing. Final molarity of the pool was determined by quantitative polymerase chain reaction using the KAPA library quantification kit (Roche). Paired-end sequencing was performed on the NextSeq 2000 (Illumina) using P3 300 cycles kits in 2 × 150-base pair (bp) format.