scRNA-Seq of mus musculus: CD45+ cells in MC38 tumor (veh)
MC38 tumors treated with vehicle were harvested and prepared into single-cell suspensions. CD45+ immune cells were isolated through positive selection using biotin-conjugated anti-mouse CD45 antibody (BioLegend) and MojoSort Streptavidin Nanobeads. The sorted CD45+ immune cells were subsequently washed with PBS containing 0.04% BSA. Cells from the same experimental group were pooled (n=3) in equal quantities to obtain approximately 15,000 cells per group. The BD Rhapsody Express Single-Cell Analysis System (BD Biosciences) was employed for single-cell capture and cDNA reverse transcription following the manufacturers protocols. Whole-transcriptome libraries were then prepared using the BD Rhapsody Whole Transcriptome Analysis Amplification Kit (BD Biosciences). Quantification of the cDNA libraries was performed using a Qubit 4.0 fluorometer with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). The size distribution of the cDNA libraries (250-1000 bp) was assessed using the DNA 1000 assay on the Agilent 2100 system (Agilent Technologies). Sequencing was conducted on a NovaSeq 6000 (Illumina) platform with 150-bp paired-end reads.