GSM3905366: Ideopathic pulmonary disease, PBMCs, p036; Homo sapiens; RNA-Seq
PBMC samples were obtained by density centrifugation. RNA was extracted with TRIzol (Invitrogen) and was re-precipitated by sodium acetate/ethanol. RNA quality and integrity were confirmed by Nanodrop (A260/A280 ratios between 1.7 and 2.2) and Bio-Analyzer mini-gel assay (Agilent, Santa Clara, CA). All RNA samples displayed a RNA Integrity Number (RIN) > 7 were proceeded to cDNA library preparation at the Genomics Core Facility of the University of Chicago (https://fgf.uchicago.edu/). Total RNA in the amount of 1μg per sample was depleted of ribosomal RNA using the Ribo-Zero kit (Epicentre, Madison, WI). The directional (first strand) cDNA libraries were prepared following the guide of TruSeq Stranded Total RNA Sample Preparation kit. RNA was fragmented at 94°C for 6 min, followed by the first strand cDNA generation. Deoxy-UTP was incorporated in second strand synthesis in order to effectively quench the second strand during PCR amplification. After adenylation of the 3' end and ligation of adapters, fragments were selected and enriched with 10 cycles of PCR amplification. Clusters were generated by bridge amplification within paired-end flow cells using Illumina HiSeq PE Cluster Kit v4 cBot according to manufacturer's instructions (Illumina, San Diego, CA). The clusters on flow cells were then sequenced on the Illumina HiSeq4000 using HiSeq SBS Kit V4.
GEO Accession
GSM3905366