GSM7073188: ZZH20220826-0, scRNAseq; Homo sapiens; RNA-Seq
The PBMCs were isolated by density gradient centrifugation using Ficoll-Paque Plus medium (GE Healthcare) and washed with Ca/Mg-free PBS. To remove the red blood cells, 2 mL GEXSCOPE® red blood cell lysis buﬀer (RCLB, Singleron) was added at 25°C for 10 minutes. The solution was then centrifuged at 500 × g for 5 min and suspended in PBS. The blood samples were centrifuged at 400g for 5 min at 4°C, and the supernatant was discarded. After removed red blood cells, PBMCs were isolated by centrifuged at 400g for 10min at 4°C. The supernatant was discarded and the PBMCs were resuspended by phosphate buffered saline to obtain a single-cell suspension Single-cell suspensions (2×105 cells/mL) with PBS (HyClone) were loaded onto microwell chip using the Singleron Matrix® Single Cell Processing System. Barcoding Beads are subsequently collected from the microwell chip, followed by reverse transcription of the mRNA captured by the Barcoding Beads and to obtain cDNA, and PCR amplification. The amplified cDNA is then fragmented and ligated with sequencing adapters. The scRNA-seq libraries were constructed according to the protocol of the GEXSCOPE® Single Cell RNA Library Kits (Singleron) (Ref). Individual libraries were diluted to 4 nM, pooled, and sequenced on Illumina novaseq 6000 with 150 bp paired end reads.