GSM7679331: XS0311-1-Gi; Mus musculus; RNA-Seq
The tissue dissociation and preparation process involved the following steps. First, the ascites sample was filtered through sterile 40-micronstrainers and centrifuged to separate the cells from the supernatant. The cell sediment was then resuspended in PBS and treated with erythrocytelysis buffer to remove erythrocytes. Fresh tissues obtained during surgery were preserved in a tissue preservation solution and subsequently washed,minced, and digested using a tissue dissociation solution. After centrifugation, the pellet was suspended in PBS. Finally, the samples were stained with Trypan Blue to assess cell viability. For reverse transcription, amplification, and library construction, single-cell suspensions were loaded onto the microwell chip by a single-cell processing system. Barcoding     beads were used to capture mRNA fromthe cells. Reverse transcription was performed to generate cDNA,followed by PCR amplification. After that, the amplified cDNA wasfragmented and ligated with sequencing adapters. scRNA-seq libraries were prepared using a specific kit and diluted to a concentration of 4 nM.The individual libraries were pooled together and sequenced on anIllumina NovaSeq 6000 platform with paired-end reads of 150 bp.Raw reads were processed using CeleScope v1.5.2 (Singleron Biotechnologies) with default parameters to generate gene expression profiles. The R1 reads were used to extract barcodes and UMIs, which were then corrected. Adapter sequences and poly A tails were trimmed from the R2 reads, and the trimmed R2 reads were aligned against  transcriptome using STAR (v2.6.1b). Uniquely mapped reads were assigned to exons using.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.