GSM8077655: Z577; Homo sapiens; RNA-Seq
For each enrolled participant, 2mL fasting venous blood samples were collected in PAXgene Blood RNA Tubes. Total RNA was extracted using the PAXgene Blood RNA Kit (QIAGEN) and isolated from peripheral blood using TRIzol® Reagent according to the manufacturer's protocol. RNA purification, reverse transcription, library construction, and sequencing were done at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) (Illumina, San Diego, CA). A library of RNA-seq transcripts was made with 1μg of total RNA and the Illumina® Stranded mRNA Prep, Ligation from Illumina (San Diego, CA). Messenger RNA was isolated using the polyA selection method with oligo (dT) beads and subsequently fragmented using a fragmentation buffer. The resulting double-stranded cDNA was synthesized using the SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). The synthesized cDNA was then subjected to end-repair, phosphorylation, and 'A' base addition as per Illumina's library construction protocol. The libraries were size selected for cDNA target fragments of 300 bp on 2% Low Range Ultra Agarose, and then PCR amplified using Phusion DNA polymerase (NEB) for 15 PCR cycles. After measuring with Qubit 4.0, the Nova Seq 6000 sequencer was used to read the paired-end RNA-seq library (2 × 150bp read length).