Illumina HiSeq 4000 sequencing; Single-cell RNA-seq of CD4+ Treg and Tmem cells from different tissues in mouse (Chromium 10x data)
Single-cell RNA-seq of CD4+ Treg and Tmem cells from different tissues in mouse (Chromium 10x data)
All organs were taken from the same pool of mice; 16 mice in total (10 females + 6 males). Isolation of mouse CD4+ T cells: Isolation of murine leukocytes for steady-state skin dataset: To isolate leukocytes from ear tissue, ears were removed at the base, split into halves and cut into very small pieces. Tissue was digested in 3.5ml RPMI media with 0.1% BSA, 15mM Hepes, 1mg/ml collagenase D (Roche) and 450μg/ml Liberase TL (Roche) for 60 minutes at 37°C in a shaking incubator at 200rpm. Digested tissue was passed through an 18G needle to further disrupt the tissue and release cells. Cells were passed through a 70μm cell strainer, and the digestion was terminated by addition of ice-cold RPMI containing 0.1%BSA and 5mM EDTA. A three-layer (30/40/70%) Percoll density-gradient was used to enrich for the lymphocytes. Cells obtained from the digestion were layered in the 30% layer on top of the 40% and 70% layers, and centrifuged for 20 minutes at 1800rpm without brake. Cells at the 40/70% interface were collected for the subsequent analysis. Cell suspensions from spleen and bLN were prepared as described previously (Uhlig et al., 2006). Isolation of murine leukocytes for steady-state colon dataset: Colons were washed twice in RPMI media with 0.1%BSA and 5mM EDTA in a shaking incubator at 200rpm at 37°C to remove epithelial cells. The tissue was then digested for an hour in the presence of RPMI/10%FCS/15mM Hepes with 100U/ml collagenase VIII. Digestion was terminated by addition of ice-cold RPMI/10% FCS/5mM EDTA. A three-layer (30/40/70%) Percoll density-gradient was used to enrich for the lymphocytes. Cells obtained from the digestion were layered in the 30% layer on top of the 40% and 70% layers, and centrifuged for 20 minutes at 1800rpm without brake. Cells at the 40/70% interface were collected for the subsequent analysis. Cell suspensions from spleen and mLN were prepared as described previously (Uhlig et al., 2006). Droplet-based scRNA-seq datasets were produced using a Chromium system (10x Genomics), referred to as 10x. Cell populations of interest were sorted, manually counted, and their concentrations adjusted to enable the capture of ~5000 cells (except for skin Treg and Tmem, for which we aimed to capture ~300 each). The standard protocol for the 10x single cell 3' kit (V2 chemistry) was followed and each cell population loaded onto a separate chip inlet Droplet-based scRNA-seq datasets were produced using a Chromium system (10x Genomics), referred to as 10x. Cell populations of interest were sorted, manually counted, and their concentrations adjusted to enable the capture of ~5000 cells (except for skin Treg and Tmem, for which we aimed to capture ~300 each). The standard protocol for the 10x single cell 3' kit (V2 chemistry) was followed and each cell population loaded onto a separate chip inlet.
Experimental Factor: cell type
CD4-positive, CD25-positive, alpha-beta regulatory T cell
Experimental Factor: organism part
colon