GSM5990011: Wildtype and Tag OVV-treated_Cell Plex; Mus musculus; RNA-Seq
tissue dissociation to prepare single cell suspension Single cell libraries are generated using the 10X Genomics Chromium Single Cell 3' Reagent V3 Kit according to manufacturer's protocol.  Cell suspensions are first assessed with Trypan Blue using a Countess FL automated cell counter (ThermoFisher), to determine concentration, viability and the absence of clumps and debris that could interfere with single cell capture.  Cells are loaded into the Chromium Controller (10X Genomics) where they are partitioned into nanoliter-scale Gel Beads-in-emulsion with a single barcode per cell.  Reverse transcription is performed and the resulting cDNA is amplified.  The full-length amplified cDNA is used to generate gene expression libraries by enzymatic fragmentation, end-repair, a-tailing, adapter ligation, and PCR to add Illumina compatible sequencing adapters.  The resulting libraries are evaluated on D1000 screentape using a TapeStation 4200 (Agilent Technologies), and quantitated using Kapa Biosystems qPCR quantitation kit for Illumina. They are then pooled, denatured, and diluted to 300pM with 1% PhiX control library added.  The resulting pool is then loaded into the appropriate NovaSeq Reagent cartridge and sequenced on a NovaSeq6000 following the manufacturer's recommended protocol (Illumina Inc.). 10X Genomics Chromium Single Cell 3' Reagent V3 Kit