title: 18005_cell28
organism: Mus musculus
description: 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).
attributes.tag: Cd4 FACS measurement
attributes.value: 4001.37687116155
attributes.tag: Cd44 FACS measurement
attributes.value: 1616.64349731788
attributes.tag: DAPI FACS measurement
attributes.value: -160.774373374729
attributes.tag: ENA first public
attributes.value: 2018-08-06
attributes.tag: ENA last update
attributes.value: 2017-09-27
attributes.tag: ERCC measurement (percent)
attributes.value: 1.7682403636
attributes.tag: External Id
attributes.value: SAMEA104323873
attributes.tag: Foxp3-GFP FACS measurement
attributes.value: 3552
attributes.tag: INSDC center alias
attributes.value: Wellcome Trust Sanger Institute
attributes.tag: INSDC center name
attributes.value: Wellcome Trust Sanger Institute
attributes.tag: INSDC first public
attributes.value: 2018-08-06T17:03:13Z
attributes.tag: INSDC last update
attributes.value: 2017-09-27T18:13:43Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Il1rl1 FACS measurement
attributes.value: 145.220025873388
attributes.tag: Itgae FACS measurement
attributes.value: 180.18218644498
attributes.tag: Klrg1 FACS measurement
attributes.value: -0.686337011894537
attributes.tag: Sell FACS measurement
attributes.value: 311.647198051166
attributes.tag: Submitter Id
attributes.value: E-MTAB-6072:18005_cell28
attributes.tag: Trbc1 FACS measurement
attributes.value: 2484.90014648438
attributes.tag: age
attributes.value: 6 to 14
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: CD4-positive, CD25-positive, alpha-beta regulatory T cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: disease
attributes.value: normal
attributes.tag: forward scatter FACS measurement
attributes.value: 83904.921875
attributes.tag: genotype
attributes.value: Foxp3-IRES-GFP
attributes.tag: individual
attributes.value: not available
attributes.tag: library
attributes.value: 3
attributes.tag: library date
attributes.value: 10/06/2015
attributes.tag: organism part
attributes.value: mesenteric lymph node
attributes.tag: phenotype
attributes.value: TCRb-positive, Foxp3-positive
attributes.tag: plate
attributes.value: 9
attributes.tag: position in library
attributes.value: treg3_091015_D4
attributes.tag: position in smart-seq2
attributes.value: C4
attributes.tag: post analysis well quality
attributes.value: true
attributes.tag: sample name
attributes.value: E-MTAB-6072:18005_cell28
attributes.tag: sex
attributes.value: not available
attributes.tag: side scatter FACS measurement
attributes.value: 29245
attributes.tag: smart-seq2 date
attributes.value: 21/09/2015
attributes.tag: strain
attributes.value: C57BL/6
attributes.tag: total reads
attributes.value: 2342580
attributes.tag: unmapped reads (percent)
attributes.value: 24.7149296929