Single cell RNA sequencing of microsatellite stable colon adenocarcinoma sample

We collected primary tumor and adjacent normal tissue from colon adenocarcinoma patients at the National Cheng Kung University Hospital (NCKUH) for single cell RNA seq analysis. Llibraries were prepared using a Chromium Single Cell 3′ Reagent Kits v3.1 (10x Genomics, Pleasanton, CA, USA). The targeted recovery was approximately 7,000 cells per sample, with an estimated multiplet rate of 6%. Fresh single-cell suspensions were processed using a Chromium Single Cell 3′ v3 platform (10x Genomics, Pleasanton, CA, USA). Sequencing libraries were prepared and sequenced on a NovaSeq 6000 system (Illumina). Base calls were converted to FASTQ files by using bcl2fastq2 (v2.20, Illumina). Read quality was evaluated using FastQC (v0.11.9; https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Processed reads were aligned to the GRCh38 reference genome and quantified using a Cell Ranger pipeline (10x Genomics; https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/). The raw gene expression matrix was filtered and normalized using the Seurat R package (version 4.3.0).