22167_1_cell229
Homo sapiens
Protocols: Isolation of human CD4+ T cells: Isolation of leukocytes from Human skin: Plastic surgery skin included reticular dermis to the depth of the fat layer. The upper 200 microns of skin were harvested using a split skin graft knife. Whole skin was digested in RF10 with 1.6mg/ml type IV collagenase for 12-16 hours at 37°C and 5%CO2. Digest was passed repeatedly through a 10ml pipette until no visible material remained. To yield a single cell suspension, digest was passed through a 100-micron filter into a polypropylene sorting tube. Wells were washed twice using cold sort buffer without calcium or magnesium to collect residual and adherent cells.Isolation of leukocytes from Human colon:Normal regions of bowel adjacent to resected colorectal tumours were prepared as previously described, with minor modifications (Bettelli et al., 2006; Geremia et al., 2011). In brief, mucosa was dissected and washed in 1 mM dithiothreitol (DTT) solution for 15 min at room temperature to remove mucus. Specimens were then washed three times in 0.75 mM EDTA to deplete epithelial crypts and were digested for 2h in 0.1 mg/ml collagenase A solution (Roche, UK). For enrichment of mononuclear cells, digests were centrifuged for 30 min at 500g in a four-layer Percoll gradient and collected at the 40%/60% interface.Peripheral blood mononuclear cell isolation: 10ml blood from skin donors were collected into EDTA. Density centrifugation with Lymphoprep was performed according to manufacturer instructions. Recovered cells were cryopreserved by pelleting and resuspending in 1ml heat-inactivated fetal calf serum containing 10% DMSO, and storing at -80ºC. Cryovials were later thawed in water bath, then rapidly being transferred to warmed medium (RPMI 1640 with 100IU/ml penicillin, 10ug/ml streptomycin, 2mM L-glutamine, 10% heat-inactivated fetal calf serum) and filtered through a 100-micron filter. 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).
ENA first public
2018-08-06
ENA last update
2017-09-27
ERCC measurement (percent)
28.762421146
External Id
SAMEA104322786
INSDC center alias
Wellcome Trust Sanger Institute
INSDC center name
Wellcome Trust Sanger Institute
INSDC first public
2018-08-06T17:03:17Z
INSDC last update
2017-09-27T18:13:09Z
INSDC status
public
Submitter Id
E-MTAB-6072:22167_1_cell229
age
not available
broker name
ArrayExpress
cell type
CD4-positive, CD25-positive, alpha-beta regulatory T cell
common name
human
disease
normal
individual
skin_3
library
1
library date
02/05/2017
organism part
blood
plate
746
position in library
J13
position in smart-seq2
E7
post analysis well quality
true
sample name
E-MTAB-6072:22167_1_cell229
sex
female
smart-seq2 date
11/06/2016
total reads
561541
unmapped reads (percent)
28.6062816428