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| Status |
Public on Sep 04, 2014 |
| Title |
mhcIIhi4wk-wildtype-cell_A02 |
| Sample type |
SRA |
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| Source name |
Mature, MHCII-high medullary thymic epithelial cell
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| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6
age: 4 weeks
aire locus: wildtype
tissue: thymus
cell type: medullary thymic epithelial cell
facs profile: CD45- EpCAM+ MHCIIhi Ly51- UEA+
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| Treatment protocol |
None.
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| Growth protocol |
C57BL/6 mice were obtained from Janvier (St. Berthevin, France).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Thymic stromal cells were isolated and sorted according to their cell surface phenotypes. Fragmented thymi were digested repeatedly for 15-20 min at 37 °C with 1mg/ml Collagenase/Dispase (Roche Diagnostic) and 100µg/ml DNaseI (Roche Diagnostic) in HBSS containing 2% FCS (Perbio), to obtain single cell suspensions. After the final digest, cells were pooled and labelled with biotinylated anti-EpCAM for positive enrichment by AutoMACS system (Miltenyi Biotec), and stained using the following directly labelled antibodies and reagents: FITC-anti-IAb (clone AF6-120.1, BioLegend), PE-anti-Ly51 (clone 6C3, BioLegend), PECy7-anti-CD45 (clone 30-F11, BioLegend), APC-Cy7-anti-I-A/E (clone M5/114.15.2, BioLegend), biotinylated anti-EpCAM (clone G8.8, DSHB, University of Iowa), Streptavidin-labelled PerCP-Cy5.5 (BioLegend) and Cy5-UEA1 (Vector Laboratories). The cells were exposed to 4’, 6-diamidino-2-phenylindole (DAPI) to identify dead cells and then sorted by flow cytometry (FACSAira, BD Biosciences). Mature, MHCII-high mTEC were sorted into media+FCS and stored on ice for 2-3 hours before Fluidigm C1 analysis.
Single cells were isolated and amplified on the Fluidigm C1 platform at the Sanger Institute/EBI Single Cell Genomics Centre. Briefly, a pool of FACS-sorted TECs (220K cells/ml) were loaded on two (denoted by the prefixes "A" and "B") medium chips (designed for capture of cells between 10-17 µM in diameter). After loading and capture, chips were inspected under the microscope and the presence of a single cell at capture sites was confirmed. Cell lysis, cDNA synthesis and subsequent PCR amplification was performed using the SMARTer Ultra Low RNA Kit (Clontech) for the C1 platform as per the manufacturer’s instructions (Clontech/Fluidigm). The cell lysis buffer (20 µL) was supplemented with 1 µL of a 1:400 dilution of the External RNA Controls Consortium (ERCC) spike-in Mix 1 (Invitrogen). Indexed Libraries from the 96 samples harvested from each chip were prepared using the Nextera XT kit (Illumina). Multiplexed libraries from each chip were sequenced over two lanes on a HiSeq 2500 (Illumina) at the Wellcome Trust Sanger Institute run in fast mode to generate 100bp paired-end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
mtec_12027_R2
PolyA RNA
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| Data processing |
(1) Alignment: Reads were aligned using the GSNAP (version 2014-01-21) algorithm with the parameters “ -N1 --maxsearch=1000 -w 500000 --pairexpect=100 --pairdev=200 -n100” to a custom genome based on the mm10 version of the mouse genome and containing the ERCC spike-in sequences.
(2) Expression quantitation: Gene expression was quantitated separately for each cell using Cufflinks (version 2.1.1) against ENSEMBL 74 reference annotations with the parameters “ --multi-read-correct --max-intron-length 500000 --library-type=fr-unstranded --frag-bias-correct --no-effective-length-correction --max-bundle-length 10000000 --max-bundle-frags 2000000 --min-intron-length 50 --max-mle-iterations 10000”
(3) Copy numbers were calculated for protein-coding genes based on per-cell normalisation curves determined from known spike-in copy number and observed spike-in FPKMs. The curves were obtained from a first order polynomial linear model forced through the plot origin for log 10 (x+1) transformed data.
(4) Cells were filtered to retain only those expressing 3,000-10,000 protein-coding genes.
Genome_build: GRCm38 (mm10)
Supplementary_files_format_and_content: A tab-delimited text file containing per-cell copy number for protein-coding genes (rows) for each single cell (columns).
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| Submission date |
Aug 11, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Stephen Sansom |
| E-mail(s) |
stephen.sansom@kennedy.ox.ac.uk
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| Organization name |
Kennedy Institute of Rheumatology
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| Department |
NDORMS
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| Lab |
Sansom
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| Street address |
Roosevelt Drive
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| City |
Oxford |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 7FY |
| Country |
United Kingdom |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE53111 |
Aire secures the transcription of polycomb marked genes to complete a comprehensive program of promiscuous gene expression in the thymic epithelial cell lineage |
| GSE60297 |
Single cell RNA-seq analysis of mature thymic epithelial cells |
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| Relations |
| BioSample |
SAMN02981037 |
| SRA |
SRX674337 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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