GSM7774397: 4NT; Homo sapiens; RNA-Seq
Inclusion criteria for patients comprised the absence of apparent metastases, a singular tumor of dimensions between 3 and 5 cm, and no prior therapeutic intervention. Ten independent surgical resected specimens were collected from five HCC patients. The collected samples were rapidly stored in sCelLiveTM Tissue Preservation Solution (Singleron) after isolation at low temperature, followed by washing with normal saline to remove blood and impurities. Following surgical excision, fresh tissues were stored in the sCelLiveTMTissue Preservation Solution (Singleron) on ice for 30 minutes. The specimens were thoroughly washed three times with Hank's Balanced Salt Solution (HBSS), then minced into small pieces, and subjected to enzymatic digestion with 3 mL of sCelLiveTM Tissue Dissociation Solution (Singleron) using the Singleron PythoN™ Tissue Dissociation System (Singleron) at 37°C for 15 minutes. The resulting cell suspension was collected and filtered through a 40-micron sterile strainer and incubated with GEXSCOPE® red blood cell lysis buffer (RCLB, Singleron) for 5-8 minutes at room temperature with a Cell: RCLB ratio of 1:2 (volume ratio) to eliminate red blood cells. The mixture was then centrifuged at 300× g at 4 ℃ for 5 minutes and the supernatant was removed, leaving behind a gentle PBS 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. Raw reads from scRNA-seq were processed to generate gene expression matrixes using CeleScope(https://github.com/singleron-RD/CeleScope) v1.9.0 pipeline. Briefly, raw reads were first processed with CeleScope to remove low quality reads with Cutadapt v1.17 to trim poly-A tail and adapter sequences. Cell barcode and UMI were extracted. After that, we used STAR v2.6.1a to map reads to the reference genome GRCh38 (ensembl version 92 annotation. UMI counts and gene counts of each cell were acquired with featureCounts v2.0.1 software, and used to generate expression matrix files for subsequent analysis.