# characteristics.cell type[].text
CD4-positive, CD25-positive, alpha-beta regulatory T cell

# characteristics.description[].text
Protocols: 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). Mouse and human cell suspensions were stained with the antibodies in Supplementary Table 1 and DAPI. Single-cells were sorted in 2μl of Lysis Buffer (1:20 solution of RNase Inhibitor (Clontech, cat. no. 2313A) in 0.2% v/v Triton X-100 (Sigma-Aldrich, cat. no. T9284)) in 96 well plates, spun down and immediately frozen at -80 degrees. Smart-seq2 protocol was largely followed to obtain mRNA libraries from single-cells. Oligo-dT primer, dNTPs (ThermoFisher, cat. no. 10319879) and ERCC RNA Spike-In Mix (1:50,000,000 final dilution, Ambion, cat. no. 4456740) were then added. Reverse Transcription and PCR were performed as previously published21, using 50U of SMARTScribe™ Reverse Transcriptase (Clontech, cat. no. 639538). The cDNA libraries for sequencing were prepared using Nextera XT DNA Sample Preparation Kit (Illumina, cat. no. FC-131-1096), according to the protocol supplied by Fluidigm (PN 100-5950 B1).