GSM8208456: IEL_KO_HTO; Mus musculus; OTHER
Single cells were prepared from the small intestine. The cells were subsequently stained with fluorochrome-conjugated monoclonal antibodies recognizing CD45 (eBioscience, clone 30-F11), DAPI for cell viability, TruStain FcX Block (BioLegend, cat 101320), 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). 70000 CD45+ and 30000 CD45- were sorted of each sample using FACSAria™ II and FACSAria™ III cell sorters (BD Biosciences) and afterwards combined to ensure enough of both CD45+ and CD45- cells would be available for sequencing. Three biological replicates were multiplexed per lane using TotalSeq-A cell hashing antibodies. Sorted IET single-cell suspensions were resuspended at an estimated final concentration of 1000 cells/µl and 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 (Beckman Coulter, B23318) 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). 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.