Single-cell RNA-seq of intestinal γδ T cells from cIAP2-deficient and RORγt-Cre–mediated cIAP1 conditional knockout mice across developmental stages
Single-cell RNA-seq of intestinal γδ T cells from cIAP2-deficient and RORγt-Cre–mediated cIAP1 conditional knockout mice across developmental stages
Small intestines and colons were dissected from adult mice and were flushed with 20 mL HBSS-HEPES to remove intestinal contents. Fat and Peyer's patches were removed, the tissues were opened longitudinally and cut into small pieces of approximately 1 cm. Tissue was washed 4 times in HBSS-EDTA buffer at 37°C while shaking at 400 rpm and then digested using 60 µg/ml LiberaseTM (Roche) and 40 µg/ml DNase (Sigma-Aldrich) per sample in supplemented RPMI for 45 minutes at 37°C on a magnetic stirrer (800 rpm). The resulting cell suspensions were filtered through 70 μm cell strainers and washed once in supplemented RPMI. Lymphocytes were separated on a 40/70 Percoll gradient. Cells from the interphase were collected and washed once before use. For neonatal gut samples, the Percoll gradient was omitted. Cells from Cre- and Cre+ RORγt-CRE-cIAP1F/F-cIAP2KO were stained in cold PBS containing bovine serum albumin (0.5%) and sorted using a BD FACSAria Fusion III. Each genotype was differentially hashed with TotalSeq anti-mouse antibodies (C0304, C0305; 1 µg per million cells; Biolegend). RNA and cDNA was isolated and prepared following the Chromium Next GEM Single Cell 5' v2 (Dual Index) User Guide (version F) from 10X Genomics. Libraries were prepared following the Chromium Next GEM Single Cell 5' v2 (Dual Index) User Guide (version F) from 10X Genomics. Library quality control and quantification was performed using a 2100 Bioanalyzer equipped with a High Sensitivity DNA kit (Agilent, 5067-4626). 