title: GSM2797474: 56_ACTGAT; Homo sapiens; RNA-Seq
library_construction_protocol: Samples were collected on day one before mechanical circulatory support surgery. Eight ml of blood was drawn into a CPT tube (Becton Dickinson, Franklin Lakes, NJ). Peripheral Blood Mononuclear cells (PBMC) from each sample were purified within 2h of phlebotomy. The collected blood was mixed and centrifuged at room temperature (22⁰C) for 20min at 3000 RPM. The cell layer was collected, transferred to 15ml conical tubes and centrifuged for 20min at 1135 RPM at 4°C. The supernatant was aspirated and discharged. The cell pellet was resuspend in cold Phosphate Buffer Saline (Sigma-Aldrich, St. Louis, MO), transferred into an eppendorf tube and centrifuged for 20min at 5.6 RPM at 4°C. The supernatant was discharged. The pellet was resuspended in 0.5 ml RNA Protect Cell Reagent (Qiagen, Valencia, CA) and frozen at -80°C. The RNA was isolated from the PBMC using RNeasy Mini Kit (Qiagen, Valencia, CA).  The quality of the total RNA was assessed using NanoDrop® ND-8000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA)  concentration above 50 ng/µl., purity - 260/280 ~ 2.0., integrity - RIN > 9.0 and average > 9.5. mRNA library was prepared with Illumina TruSeq RNA kit according to the manufacturer’s instructions (Illumina, San Diego, CA). Library construction consists of random fragmentation of the polyA mRNA, followed by cDNA production using random polymers. The cDNA libraries were quantitated using Qubit and size distribution was checked on Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).
attributes.tag: GEO Accession
attributes.value: GSM2797474