title: 22379_3_cell40
organism: Homo sapiens
description: 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).
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: 28.627815127
attributes.tag: External Id
attributes.value: SAMEA104320912
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: 2017-09-27T18:12:07Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6072:22379_3_cell40
attributes.tag: age
attributes.value: not available
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: central memory CD4-positive, alpha-beta T cell
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: normal
attributes.tag: individual
attributes.value: skin_1
attributes.tag: library
attributes.value: 3
attributes.tag: library date
attributes.value: 11/11/2015
attributes.tag: organism part
attributes.value: skin
attributes.tag: plate
attributes.value: plate_skin_7
attributes.tag: position in library
attributes.value: H5
attributes.tag: position in smart-seq2
attributes.value: H5
attributes.tag: post analysis well quality
attributes.value: true
attributes.tag: sample name
attributes.value: E-MTAB-6072:22379_3_cell40
attributes.tag: sex
attributes.value: female
attributes.tag: smart-seq2 date
attributes.value: 28/10/2015
attributes.tag: total reads
attributes.value: 677450
attributes.tag: unmapped reads (percent)
attributes.value: 54.1229611041