GSM8896959: MC38 tumor, KO mouse; Mus musculus; RNA-Seq
MC38 tumors from wild-type and LMO7 knockout mice were isolated, prepared into single-cell suspensions using the Miltenyi Biotec gentleMACS Octo Dissociator with Heaters, following the 37C-m-TDK-1 program (Miltenyi Biotec), and passing through 70 µm cell strainers. After red blood cell (RBC) lysis, approximately 10,000 FACS-sorted live cells (1,000 cells/µl suspension) were resuspended in 1x PBS (calcium- and magnesium-free) containing 0.04% BSA (A2153, SIGMA). The cell suspension was used as input in 10x Chromium Controller System (10x Genomics Inc., product code 120223), and cell barcoding was performed using gel beads in emulsion (GEMs) in assembly chip. GEM reverse transcription (RT) reactions were conducted in a thermocycler at 53°C for 45 minutes, followed by 85°C for 5 minutes, and held at 4°C overnight. SPRIselect dynabeads (B23318, Beckman Coulter) were utilized for GEM recovery after RT. DNA quantities ranging from 2 to 50 ng were used for target enrichment. cDNA amplification, fragmentation, end repair, and A-tailing preparation were performed according to the manufacturer's protocol. Quality control and quantification of the gene expression library were conducted at various stages using high-sensitivity DNA chips and an Agilent 2100 Bioanalyzer (Agilent Technologies). Quality control was performed twice prior to sequencing. Two barcoded scRNA samples were pooled together before sequencing, which was performed on the NovaSeq 6000 S1 platform with a sequencing depth of approximately 300 million reads.