GSM8697908: HC_16_48, bulk RNA; Homo sapiens; RNA-Seq
Peripheral blood samples were collected and stored at 4℃ in EDTA anticoagulant tubes and processed promptly. PBMC isolation was performed using a Ficoll density gradient centrifugation with the following steps: whole blood samples were diluted 1:1 with phosphate buffered saline (PBS), and the diluted blood samples were gently layered over Ficoll solution in a centrifuge tube. The mixture was centrifuged at 400g for 30 minutes at room temperature, with the acceleration and brake settings of the centrifuge set to 1. After centrifugation, the intermediate cloudy layer containing the PBMCs was carefully collected. This layer was mixed with three volumes of PBS and centrifuged again at 500g for 10 minutes at room temperature. The resulting PBMC pellet was washed further with PBS and centrifuged to enrich the cells. Samples with significant red blood cell contamination were treated with ACK Lysing Buffer (A10492-01, Gibco) for red blood cell lysis. For PBMCs sequencing, the total RNA was mixed with ERCC RNA Spike-in Mix (4456740, Invitrogen) in proportion for transcriptome library construction. The sequencing library was prepared using the RNA-seq Library Prep Kit for MGI (NRM605, Vazyme) and sequenced in batches on the DNBSEQ-T7 platform.