GSM8552759: WT; Mus musculus; RNA-Seq
CD45+ cells were sorted from tumors and single cell libraries were generated using the 10X Genomics platform.  First, Feature Barcode conjugated molecules were bound to cell surface proteins.  Then live-CD11b+ cells were isolated using the SONY MA900 cell sorter (Sony Biotechnology).  Single-cell suspensions were pooled together and assessed with AOPI stain using a Cellometer K2 automated cell counter (Nexcelom), to determine concentration, viability and the absence of clumps and debris that could interfere with single cell capture.  Cells were loaded into the Chromium Controller (10X Genomics) where they were partitioned into nanoliter-scale Gel Beads-in-emulsion with a single barcode per cell.  Reverse transcription was performed, and the resulting cDNA is amplified. Amplified cDNA is separated into full-length and Feature Barcode fractions using SPRISelect beads (Beckman Coulter).  The full-length amplified cDNA was used to generate libraries by enzymatic fragmentation, end-repair, a-tailing, adapter ligation, and PCR to add Illumina compatible sequencing adapters.  Feature barcode derived cDNA was PCR amplified to incorporate Illumina adapter sequences and unique sample indexes.  The resulting libraries were evaluated on D1000 screentape using a TapeStation 4200 (Agilent Technologies) and quantitated using Kapa Biosystems qPCR quantitation kit for Illumina.  They were then pooled, denatured, and diluted to 300 pM with 1% PhiX control library added.  The resulting pool was then loaded into the appropriate NovaSeq Reagent cartridge and sequenced on a NovaSeq6000 following the manufacturer's recommended protocol (Illumina Inc.).