GSM8129012: Ripk1/Casp8 KO, ADT; Mus musculus; OTHER
Half of the cells were used for enrichment of T cells using the MagniSort™ Mouse T cell Enrichment Kit (8804-6820-74, eBioscience) according to the manufacturer's instructions. The other half of the single cell suspensions were not enriched. Cells were subsequently stained with fluorochrome-conjugated monoclonal antibodies recognizing CD4 (clone RM4-5, BD Biosciences) and CD8β (clone YTS156.7.7, Biolegend), DAPI for cell viability, TruStain FcX Block (101320, BioLegend), the mouse cell surface protein antibody panel containing 165 oligo-conjugated antibodies and 9 TotalSeq-A isotype controls (TotalSeq-A, BioLegend) and a unique TotalSeq-A cell hashing antibody (BioLegend). Sorting of live CD4+ and CD8+ T cells from the enriched cells and sorting of total live cells from the unenriched cells was performed using FACSAria™ II and FACSAria™ III cell sorters (BD Biosciences). This resulted in two suspension of sorted cells per mouse which were tagged with unique hashing antibodies and combined in a 1:1 ratio to ensure enough T cells would be available for sequencing as T cell numbers are significantly reduced in the mLN of Ripk1ΔCD4 mice. Three biological replicates per sample were loaded (consisting of a 1:1 combination of total live cells and enriched T cells) were multiplexed per lane using TotalSeq-A cell hashing antibodies. In the final analysis, only two of the three Ripk1FL/FLCasp8FL/FL samples had sufficient cells to analyse. Sorted single-cell suspensions at an estimated final concentration of 1500 cells/µl were loaded on a Chromium GemCode Single Cell Instrument (10x Genomics) to generate single-cell gel beads-in-emulsion (GEM). The scRNA-Seq libraries were prepared using the GemCode Single Cell 3' Gel Bead and Library kit, version 3.1 (10x Genomics) according to the manufacturer's instructions with the addition of amplification primers (3nM each, 5'CCTTGGCACCCGAGAATT*C*C - 5'GTGACTGGAGTTCAGACGTGTGC*T*C) during cDNA amplification to enrich the TotalSeq-A cell surface and hashing protein oligos, respectively. Size selection with SPRIselect Reagent Kit (B23318, Beckman Coulter, Brea, CA, US) was used to separate amplified cDNA molecules for 3' gene expression and cell surface protein construction. TotalSeq-A protein library construction including sample index PCR using Illumina's Truseq Small RNA primer sets and SPRIselect size selection was performed according to the manufacturer's instructions. The cDNA content of pre-fragmentation and post-sample index PCR samples was analyzed using the 2100 BioAnalyzer (Agilent, Santa Clara, CA, US). Sequencing libraries were loaded on an Illumina NovaSeq flow cell at VIB Nucleomics core with sequencing settings according to the recommendations of 10x Genomics, pooled in a 70:20:10 ratio for the combined 3' gene expression, cell surface and hashing protein samples, respectively.