GSM5370808: sample.0205.t0; Homo sapiens; RNA-Seq
Whole peripheral blood was collected at both visits, t0 and t1, at the Pain Service of University Hospital of Parma. Tempus blood RNA tubes were used (Applied Biosystems, n. 4342792, Beverly, MA, USA), shaken vigorously for 10–15 sec immediately after collection for RNA stabilization, and stored at -20 C. Total RNA was isolated using Maxwell® 16 LEV simplyRNA Blood Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. After, total RNA was quantified using the NanoDrop ND-1000 (Thermo Scientific, Waltham, MA, USA), and optical purity of RNA was defined according to the 260/280 ratio not less than 1.8 and 260/230 ratio between 1.8–2.2 of the isolated RNA. Then, the RNA integrity was assessed with the Agilent 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA) using the RNA Screen Tape assay (Agilent Technologies, n. 5067-5576). An average of 8 µg of total RNA was recovered from frozen whole blood, displaying RIN values between 6.5–8.4. Purified RNA was stored at -80 °C in 25-µl aliquots. All RNA-sequencing was performed by the Genome Quebec Innovation Centre (McGill University, Montreal, Canada). Transcriptome libraries were generated from 1 μg of total RNA using the Kapa RNA-stranded Sample Prep Kit (KK8400, KAPABiosystems, Wilmington, MA, USA) following the manufacturer's protocols. Briefly, poly-A mRNA was purified using poly-T oligo-attached magnetic beads using two rounds of purification. During the second elution of the poly-A RNA, the RNA was fragmented and primed for cDNA synthesis. During cDNA synthesis, dUTP was incorporated in the second-strand synthesis, and subsequently the dUTP-containing strand was selectively degraded. Adenylation of the 3' ends and ligation of adapters were done following the manufacturer's protocol. Enrichment of DNA fragments with adapter molecules on both ends was performed using 10 cycles of PCR amplification using the KAPA PCR mix and Illumina-adapted primers cocktail. Paired-end 2x100 nucleotides sequencing was performed using the Illumina HiSeq2000 machine running TruSeq v3 chemistry at Genome Québec.
GEO Accession
GSM5370808