Single-cell resolution spatial analysis of antigen-presenting cancer-associated fibroblast niches [scRNA-seq]

To understand the microenvironmental changes caused by Spp1 knockout, we digested the tumors from the control and knockout groups into single cell suspension and performed scRNA-seq. The scRNA-seq data suggested a significant increase of T cell infiltration into the stromal Spp1 knockout tumors. For each group, we included 6 tumors. Every two tumors were then pooled together for library construction (three libraries per group). Approximately 10,000 cells were used for library construction with the Chromium Next GEM Single Cell 3′ Kit v3.1 (10x Genomics), following the manufacturer’s protocol. The purified libraries were sequenced on an Illumina NovaSeq 6000. Raw base call (BCL) files from the sequencer were demultiplexed into fastq files. These fastq files were aligned to the mm10 mouse reference genome and quantified using the Cell Ranger pipeline (version 7.2.0, 10x Genomics). The preliminary filtered data generated from Cell Ranger were utilized for all subsequent analyses.