GSM8848791: Tumor_LN_Cre_WT; Mus musculus; RNA-Seq
tumors were excised, minced with a razor blade, and digested for 30 minutes in DMEM containing 10 mM HEPES, 1.6 mg/mL collagenase IV (Sigma, #C5138), and 10 μg/mL DNase I (Sigma, #4942078001). The resulting cell suspensions were filtered through a 40 μm mesh, and samples from two mice were pooled for each experiment. After red blood cell lysis (Gibco, #A10492-01), cells were stained with Zombie Aqua viability dye (BioLegend, #423105) and a FITC anti-mouse CD45 antibody (BioLegend, #103107). Approximately 500,000 live immune cells, which included ~80% CD45+ cells, were sorted on a BD FACSAria III cell sorter into a collection buffer (PBS, 0.04% BSA, and 50% FBS; Sigma, #F4135), and then fixed. Single-cell RNA sequencing (scRNA-seq) libraries were prepared using the Chromium 10x Fixed RNA kits (#PN-1000414 and #PN-1000497) following the manufacturer's protocol. A total of eight Cre (knockout) and eight WT (wild-type) tumor-infiltrating immune cell and lymph nodes samples were uniquely barcoded during library preparation. For tumor-infiltrating immune cell, the library included Tumor_Cre_rep1, Tumor_Cre_rep2, Tumor_Cre_rep3, Tumor_Cre_rep4, Tumor_WT_rep1, Tumor_WT_rep2, Tumor_WT_rep3, and Tumor_WT_rep4. Following barcoding, all samples were pooled into a single tube and sequenced on an Illumina NovaSeq platform using paired-end sequencing. Sequencing was performed across seven lanes (L001 to L007), generating two FASTQ files per lane (one Read 1 [R1] and one Read 2 [R2]), resulting in a total of 14 FASTQ files.  Post-sequencing, raw FASTQ files were processed using the Cell Ranger pipeline for demultiplexing and alignment. Each of the eight Cre and eight WT samples has separate processed output files, including a matrix file (.mtx.gz), a barcode file (.tsv.gz), and a feature file (.tsv.gz). The processed data files for each sample are named accordingly, such as Tumor_Cre_rep1_barcodes.tsv.gz, Tumor_Cre_rep1_features.tsv.gz, and Tumor_Cre_rep1_matrix.mtx.gz for Tumor_Cre_rep1, with similar naming conventions for all other samples.