GSM6185956: Il1rl1-ExAB-/- (ADT, HTO); Mus musculus; OTHER
Wildtype and Il1rl1-ExAB-/- mice (n = 3 per genotype) were infected intravenously with LCMV-WE (2x106 PFU). At day 7 post infection, splenocytes were isolated by mechanical disruption and erythrocytes were lysed by incubation in erythrocyte lysis buffer (10 mM KHCO3, 155 mM NH4Cl, 0.1 mM EDTA, pH 7.5). Obtained cell suspensions were passed through 70µm filters and T cells were enriched by depleting CD19+ B cells using magnetic activated cell sorting (MACS, Miltenyi Biotec). Subsequently, live CD90+ CD8+ CD44+ T cells were flowcytometrically sorted using a BD Aria II device, stained with Total-Seq antibodies (Biolegend) and LCMV dCODE dextramers (Immudex) according to manufacturer's instructions and subjected to the 10x Genomics scRNA-Seq workflow. See sorting strategy. 10x Genomics workflow for cell capturing. For the preparation of scRNA gene expression (GEX), TCR and CiteSeq libraries the Chromium Next GEM Single Cell 5' Library & Gel Bead Kit v1.1 as well as the Chromium Single Cell 5' Feature Barcode Library Kit (10x Genomics) were used for CD127 (GTGTGAGGCACTCTT), antiAPC (TTAACCGTCTCCCTT), CD279 (GAAAGTCAAAGCACT), CD197 (TTATTAACAGCCCAC), CD39 (GCGTATTTAACCCGT), CD185 (ACGTAGTCACCTAGT), CD31 (GCTGTAGTATCATGT), KLRG1 (GTAGTAGGCTAGACC), CD183 (GTTCACGCCGTGTTA), CD49d (CGCTTGGACGCTTAA), CD73 (ACACTTAACGTCTGG), FQPQNGQFI (GTGGTCGACCGTCTG), KAVYNFATC (CCTTAATATGTAGAA), SGVENPGGYCL (AGGAACCGCTGTTGA), CSANNSHHYI (TCATGTCAGACTTGG), ISHNFCNL (CGTGCAAGGCCGGAA) as well as , Hashtag1 (ACCCACCAGTAAGAC), Hashtag2 (GGTCGAGAGCATTCA), Hashtag3 (CTTGCCGCATGTCAT), Hashtag4 (AAAGCATTCTTCACG),Hashtag5 (CTTTGTCTTTGTGAG). After cDNA amplification the feature barcode libraries were prepared separately using the Single Index Kit N Set A (10x Genomics). TCR target enrichment was performed using the Chromium Single Cell V(D)J Enrichment Kit for murine T cells (10x Genomics). Final GEX and TCR libraries were obtained after fragmentation, adapter ligation and final Index PCR using the Single Index Kit T Set A (10x Genomics). The Qubit dsDNA HS assay kit (life technologies) was used for library quantification and fragment sizes were determined using the Fragment Analyzer with the NGS Fragment Kit (1-6000bp) (Agilent) Chromium Next GEM Single Cell 5' Library & Gel Bead Kit v1.1 as well as the Chromium Single Cell 5' Feature Barcode Library Kit (10x Genomics) were used