18005_cell140
Mus musculus
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).
Cd4 FACS measurement
2020.85931675524
Cd44 FACS measurement
1152.91332229365
DAPI FACS measurement
-200.079952638878
ENA first public
2018-08-06
ENA last update
2017-09-27
ERCC measurement (percent)
1.7694508662
External Id
SAMEA104323874
Foxp3-GFP FACS measurement
9419
INSDC center alias
Wellcome Trust Sanger Institute
INSDC center name
Wellcome Trust Sanger Institute
INSDC first public
2018-08-06T17:03:13Z
INSDC last update
2017-09-27T18:13:43Z
INSDC status
public
Il1rl1 FACS measurement
204.407420374165
Itgae FACS measurement
160.326561594985
Klrg1 FACS measurement
47.2401050135783
Sell FACS measurement
439.460914835646
Submitter Id
E-MTAB-6072:18005_cell140
Trbc1 FACS measurement
3094.85009765625
age
6 to 14
broker name
ArrayExpress
cell type
CD4-positive, CD25-positive, alpha-beta regulatory T cell
common name
house mouse
disease
normal
forward scatter FACS measurement
109220.5625
genotype
Foxp3-IRES-GFP
individual
not available
library
2
library date
10/01/2015
organism part
mesenteric lymph node
phenotype
TCRb-positive, Foxp3-positive
plate
9
position in library
treg2_091015_D6
position in smart-seq2
B6
post analysis well quality
true
sample name
E-MTAB-6072:18005_cell140
sex
not available
side scatter FACS measurement
29923
smart-seq2 date
21/09/2015
strain
C57BL/6
total reads
2146242
unmapped reads (percent)
21.7270932169