GSM5579361: PBMC14_PD; Homo sapiens; RNA-Seq
Frozen PBMCs vials were rapidly thawed in a 37°C water bath for ~2 minutes, and the vials were removed when a tiny ice crystal was left. Thawed PBMCs were quenched with 13 ml 37°C pre-warmed 1X phosphate-buffered saline (PBS, Thermo Fisher Scientific, #10010031) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, #A3160601). Cells were centrifuged at 300 x g for 10 minutes at room temperature. The supernatant was removed, and cell pellet was resuspended in 3 ml 1X PBS containing 10% FBS, passed through a 40μm cell strainer (Falcon, #352340), then centrifuged at 300 x g for 10 minutes at room temperature. Dead cells were removed by magnetic beads purification (Miltenyi Biotech, #130-090-101) according to the manufacturer's protocol. Cells were resuspended with cell resuspension buffer at a concentration of 1,000 viable cells/μl. The DNBelab C Series Single-Cell Library Prep Set (MGI, #1000021082) was utilized as previously described (Liu et al., 2019). In brief, single-cell suspensions were used for droplet generation, emulsion breakage, beads collection, reverse transcription, and cDNA amplification to generate barcoded libraries. Indexed scRNA-seq libraries were constructed according to the manufacturer's protocol. The DNA nanoballs (DNBs)-based libraries were sequenced by the ultra-high-throughput DIPSEQ T1 sequencer at China National GeneBank (CNGB). The read structure was paired-end with Read 1, covering 30 bases inclusive of 10-bp cell barcode 1, 10-bp cell barcode 2 and 10-bp unique molecular identifier (UMI), and Read 2 containing 100 bases of transcript sequence, 10-bp sample index.