title: GSM7829855: SingleCell WT gene expression [WT_gex]; Mus musculus; OTHER
library_construction_protocol: MC38 tumors were pooled and digested to obtain single cell suspensions. Tumor infiltrating MDSCs were stained using antibodies CD45.1 vs CD45.2 to distinguish WT vs KO cells. Cells were also stained with antibody Derived Tag directed against surface markers CD11C, Ly6c, Ly6G . Each antibody is barcoded with a unique Oligonucleotide Hash Tag.   10e4 sorted viable cells  were loaded into a Chromium Controller (10X Genomics). Viable sorted CD11b+ cells were washed three times before being processed using 10X genomics Feature barcoding protocol and workflow with v3.1 3' technology. The single-cell RNAseq libraries are prepared by the Genomics Core facility at Lerner Research Institute. The single-cell RNAseq libraries are prepared according to the manufacturer's Chromium Single Cell 3ʹ v3.1 with Feature Barcode technology for Cell Surface Protein (CSP) protocol. Briefly,cDNA for the 3' gene expression library was initially synthesized from poly-adenylated mRNA, and DNA for the CSP library was synthesized from the cell surface protein feature Barcode. A minimum of 1 nanogram of cDNA was used to beginfragmentation, followed by end-repair, adapter ligation, and sample indexing. Each sequencing library including the CSP library was tagged with a dual index (10-nucleotide long each) sample barcode that identifies from which sample a sequence was derived. In addition, a 12-base unique molecular indexes (UMIs) were introduced to reduce duplicate reads.Constructed libraries are pooled and quantified using a Quantabio Q cycler.NovaSeq 6000RapidRun flow cells are used to sequence a minimum of 20,000 read pairs per cell for the Gene expression library and a minimum of 5,000 read pairs per cell for the Cell surface protein library using specifications recommended by the manufacturer (10X Genomics, document # CG000317).