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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 04, 2014 |
| Title |
cTEC R1 |
| Sample type |
SRA |
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| Source name |
wild type cTEC
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
aire locus: wildtype
age: 1 week
tissue: thymus
celltype: cortical thymic epithelial cell (cTEC)
facs profile: CD45- EpCAM+ MHCII+ 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). A novel mouse line, designated AireGFP/+, was generated to target the enhanced green fluorescent protein (EGFP) expression to Aire positive cells. In short, a knock-in targeting vector was used to replace the first exon of the Aire locus with the sequence of EGFP. Following 129/Ola embryonic stem cell (ESC) electroporation and screening for homologous recombinants by Southern blotting and PCR, verified ESC were used for blastocyst injections. Germ-line transmitted heterozygous mice were bred onto a C57BL/6 genetic background. Heterozygous and homozygous mice were born at the expected Mendelian frequency. The transgenic mice displayed a thymic cellularity of lymphoid and stromal cells that were indistinguishable from that of wild type mice. Mice were maintained under specific pathogen-free conditions and according to Swiss federal and institutional regulations.
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| Extracted molecule |
polyA 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) achieving a TEC purity of over 93%. Sorted TEC were pelleted, lysed in the RLT lysis buffer of the RNeasy Micro Kit (Qiagen), or cross-linked for ChIP and kept at -80 °C until use. Total RNA was isolated from sorted TEC using the RNeasy Micro Kit.
PolyA+ selected RNA-seq libraries were generated from 1 µg of total RNA by mRNA-Seq Sample Prep Kit (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
ctec1wk-wildtype-R1
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| Data processing |
(1) Alignment: 36bp single end reads were mapped to the mouse genome (mm9) using tophat (version 1.4.1) with the options “ --library-type fr-unstranded --segment-length 18” together with reference junctions and a transcriptome index.
(2) Expression quantitation: FPKM values were calculated using cuffdiff (version 2.0.2) with the options "--upper-quartile-norm --library-type=fr-unstranded --frag-len-mean=200 --frag-len-std-dev=80 --no-effective-length-correction" together with a mask file (ribosomal and mitochondrial rRNA/tRNA) and a gtf file containing the sanitised ensembl 67 reference coding geneset.
Genome_build: mm9
Supplementary_files_format_and_content: A tab-delimited text file containing cuffdiff calculated FPKM values for the seven TEC populations.
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| Submission date |
Dec 06, 2013 |
| 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 |
GPL11002 |
| Series (2) |
| GSE53110 |
Gene expression in thymic epithelial cells [RNA-seq] |
| GSE53111 |
Aire secures the transcription of polycomb marked genes to complete a comprehensive program of promiscuous gene expression in the thymic epithelial cell lineage |
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| Relations |
| Reanalyzed by |
GSE80797 |
| BioSample |
SAMN02438239 |
| SRA |
SRX388798 |
| 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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