GSM7102462: Control6; Homo sapiens; RNA-Seq
The abstraction of total RNA was isolated from the frozen tissues by TRIzol (LifeTechnology,USA) following the manufacturer's instructions. The tissue samples were placed in 2ml RNA-free EP tube and ground with four 4mm steel balls in TissueLyser LT (QIAGEN, Dusseldorf, Germany). The purity and quality of the isolated RNA of each sample were assessed by measuring the OD260/280 ratio, with values ranging from 1.8-2.1 being considered acceptable. RNA deep sequencing was carried out by Genergy (Shanghai,China), using a total of 2ug RNA to synthesize the first strand of cDNA, according to Promega GoScript's protocol (Promega,USA). Strand-specific RNA-seq Libraries were generated with TruSeq® RNA LT Sample Prep Kit v2 (Illumina, CA, USA) following the guidelines. The Ribo-Zero Gold kit (Illumina, CA, USA) was used to remove rRNA, and the remaining RNA fragments was used to synthesize double-stranded cDNA. The double-stranded cDNA products were then end-repaired, added A-tailed and ligated to adaptors. Finally, PCR amplification was performed to enrich cDNA libraries, and the quality of libraries was controlled by Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technology, MA, USA). Sequencing was performed on an Illumina Hiseq 2500 instrument (Illumina, CA, USA) with the TruSeq PE Cluster Kit v3-cBot-HS (Illumina, CA, USA).