GSM6890705: PESA Study participant mRNA  SUAZ_503_v1; Homo sapiens; RNA-Seq
Peripheral blood samples were collected from 391 PESA participants, transferred to PAXgene Blood RNA tubes (Preanalytix) to allow instant preservation of RNA, and stored at -80ºC until use. Total cellular RNA, including miRNA, was automatically extracted from PAXgene tubes using a QIAsymphony SP liquid handling robot (Qiagen) and the QIAsymphony PAXgene blood RNA kit (Qiagen), following the manufacturer´s instructions. RNA quantity was measured by absorbance using a NanoQuant Plate (Tecan). The RNA integrity number (RIN) was calculated by automated electrophoresis in a 2100 Bioanalyzer (RNA6000 Nano LabChip, Agilent). Barcoded RNA-seq libraries were generated using 200 ng of total RNA with RIN>6 and the NEBNext Ultra RNA Library preparation kit (New England Biolabs). Briefly, polyA+ RNA was purified using polyT oligo-attached magnetic beads followed by fragmentation, and subsequent first and second cDNA strand synthesis. cDNA 3' ends were adenylated, the adapters were ligated, and the library was amplified by PCR. The size and concentration of the libraries were checked using the Agilent 2100 Bioanalyzer DNA 1000 chip. Pools were prepared containing on average 11 libraries with a different barcode and equimolar concentrations of each library. Each pool concentration was determined using the Qubit® fluorometer (Life Technologies). Library pools were sequenced on a HiSeq2500 (Illumina) to generate 60 bases single reads. Fastq files for each sample were obtained using CASAVA v1.8 software (Illumina).