GSM8395973: hCiCMs_scRNA; Homo sapiens; RNA-Seq
The cells were collected by trypsinization. The ascites samples went through filtering by 40-micron sterile strainers and were  centrifuged at 350× g for 5 min. Then the supernatant was discarded, and the sediment was resuspended in 1ml PBS (HyClone). Finally, the samples were stained with Trypan Blue and the cell viability was evaluated microscopically. 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)1. Individual libraries were diluted to 4 nM, pooled, and sequenced on Illumina novaseq 6000 with 150 bp paired end reads.Raw reads were processed to generate gene expression profiles using CeleScope v1.5.2 (Singleron Biotechnologies) under default setting. Briefly, Barcodes and UMIs were extracted from R1 reads and corrected. Adapter sequences and poly A tails were trimmed from R2 reads and the trimmed R2 reads were aligned against the Homo_sapiens_ensembl_92 transcriptome using STAR(v2.6.1b). Uniquely mapped reads were then assigned to exons with FeatureCounts(v2.0.1). Successfully Assigned Reads with the same cell barcode, UMI and gene were grouped together to generate the gene expression matrix for further analysis. 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.