GSM8390319: Vessel,T48h Hb Treatment, 02; Homo sapiens; RNA-Seq
Total RNA isolation was performed using RNeasy® Mini Kit (Qiagen CatNo:74104), for which the initial sample mincing was carried out on dry ice with sterilized, pre-chilled blade and pipette tips to avoid degradation of RNA, then transferred to RLT lysis buffer containing beta-mercaptoethanol. Other than this initial set of sample processing, the rest of the steps, including DNase I treatment, were followed as instructed in the protocol. Total RNA was quality-checked using Agilent RNA 6000 Pico kit (#5067-1513) by Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA). RNA with an integrity number of greater than 7 was used for library preparation. Libraries were prepared with SMART-Seq V4 PLUS Kit (R400752, Takara Bio, Japan) following the manufacturer's instructions. The quality of the final libraries was examined using Agilent High Sensitive DNA Kit (#5067-4626) by Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA) , library concentrations were determined by qPCR using Collibri Library Quantification kit (#A38524500, Thermo Fisher Scientific). The libraries were pooled evenly and went for the paired-end 75-cycle sequencing on an Illumina NextSeq 550 System (Illumina, Inc., USA) using High Output Kit v2.5 (#20024907, Illumina, Inc., USA).