characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2017-08-01T17:01:18Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-05-16T18:32:05Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA104036971
characteristics.characteristic: External Id
characteristics.text: unspecified
characteristics.characteristic: INSDC center name
characteristics.text: 2017-08-01T17:01:18Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-05-16T18:32:05Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1700864
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-5727:exp2lib2_cell38
characteristics.characteristic: Submitter Id
characteristics.text: 4
characteristics.unit: week
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: medullary thymic epithelial cells
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0002365
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Thymi were collected from 2 and 4 week-old wild-type C57BL/6 male and female mice. Up to 3 thymi were cleaned of fat and connective tissue, finely minced and pooled together. Thymocytes were flushed by gentle agitation with a magnetic stirrer in RPMI-1640 for 30 min, at 4ºC. Thymic fragments were recovered by settling, and the supernatant discarded. After further dispersion, three additional washes were performed. Fragments were then incubated in 5 mL of 0.125% (w/v) Collagenase D and 0.1% (w/v) DNAse I (both from Roche) in RPMI-1640, at 37ºC for 15 min, with gentle pipetting every 5 min. The supernatant was collected and kept on ice, while the thymic fragments were subject to two further incubations. The remaining fragments were finally resuspended in 5ml of 0.125%(w/v) Collagenase/Dispase (Roche) and 0.1%(w/v) DNaseI in RPMI-1640 for 30 min at 37ºC, with gentle agitation every 15 min. All the collected fractions were pooled, centrifuged at 450x g for 5 min and incubated in 5 mM EDTA, 1% FCS, 0.02% (w/v) NaN3 in PBS (EDTA/FACS buffer) for 10 min at 4ºC. After filtering through a 100μm-strainer, the resulting cell suspension was depleted of hematopoietic cells by Magnetic-Activated Cell Sorting (MACS) using CD45-MicroBeads (Miltenyi Biotec). For sorting, the recovered fraction was blocked using anti-CD16/CD32 (clone 2.4G2, Tonbo) and then stained using anti-CD45-PerCP-Cy5.5 (clone 30-F11, BioLegend), anti-Ly-51-FITC (clone 6C3, BioLegend), anti-CD326(Ep-CAM)-AF647 (clone G8.8, BioLegend), UEA-1-Biotin and Streptavidin-Pacific Blue. Propidium iodide was used as a viability dye. mTECs (PI−CD45−EpCAM+Ly51−UEA-1+) were sorted with a MoFlo™ XDP (Beckman Coulter, Inc.). Single cell capture and processing for the main dataset was performed using the Fluidigm C1 system. The cell suspension obtained from sorting was loaded onto the Fluidigm C1 platform using medium-sized capture chips (10-17μm cells). 1 μl of a 1:400 or 1:2000 dilution of External RNA Control Consortium (ERCC) spike-ins (Ambion, Life Technologies) were included in the lysis buffer. Reverse transcription and cDNA preamplification were performed using the SMARTer Ultra Low RNA kit (Clontech). The cDNA libraries for sequencing were prepared using Nextera XT DNA Sample Preparation Kit (Illumina), according to the protocol supplied by Fluidigm (PN 100-5950 B1). Libraries from 96 single cells were pooled and purified using AMPure XP beads (Beckman Coulter).
characteristics.characteristic: description
characteristics.text: wild type genotype
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005168
characteristics.characteristic: genotype
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: thymus
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: C57BL/6
characteristics.characteristic: strain
characteristics.text: exp2lib2_cell38
characteristics.characteristic: title