title: GSM5425256: PBMCs, RP_6; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated and purified using TRIzol(Life, cat.265709, CA, USA) following the manufacturer's procedure. After the quality inspection of Agilent 2100 Bioanalyzer (Agilent, cat.G2939AA, CA, USA) and NanoPhotometer® (Implen, cat.N60, Munich, Germany), poly (A) RNA is purified from 1μg total RNA using VAHTS® mRNA Capture Beads with Oligo (dT) (Vazyme, cat.N401-01, Nanjing, China) using two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using VAHTS® Universal V6 RNA-seq Library Prep Kit (Vazyme, cat.NR604, Nanjing, China) under 94℃   8min. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by reverse transcription reagent, which were next used to synthesise U-labeled second-stranded DNAs. An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs, size selection was performed with VAHTS® DNA Clean Beads (Vazyme, cat. N411, Nanjing, China). Then the ligated products are amplified with PCR. At last, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 (Guangzhou Huayin Health Technology Co., Ltd. Guangzhou, China) following the vendor's recommended protocol.