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Sample GSM1282515 Query DataSets for GSM1282515
Status Public on Sep 04, 2014
Title mature mTEC R1
Sample type SRA
 
Source name wild type mature mTEC
Organism Mus musculus
Characteristics strain background: C57BL/6
aire locus: wildtype
age: 4 weeks
tissue: thymus
celltype: medullary thymic epithelial cell (mTEC)
facs profile: CD45- EpCAM+ MHCIIhi Ly51- UEA+
Treatment protocol None.
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.
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).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer IIx
 
Description mhcIIhi4wk-wildtype-R1
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.
 
Submission date Dec 06, 2013
Last update date May 15, 2019
Contact name Stephen Sansom
E-mail(s) stephen.sansom@kennedy.ox.ac.uk
Organization name Kennedy Institute of Rheumatology
Department NDORMS
Lab Sansom
Street address Roosevelt Drive
City Oxford
State/province Oxfordshire
ZIP/Postal code OX3 7FY
Country United Kingdom
 
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
Relations
Reanalyzed by GSE80797
BioSample SAMN02438242
SRA SRX388806

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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