Dataset Information

Gene expression profiling of perinatal or adult medullary thymic epithelial cells (MECs)

ABSTRACT: A comparative analysis of gene expression of perinate or adult Aire-GFP+ and GFP- MECs in WT or Aire-KO thymus Aire+ and Aire- MECs were sorted from adults (5 weeks old) or perinates (0-3 days old) of Aire-driven Igrp-GFP (Adig) mice in Aire-WT and -KO background. RNA from whole samples was amplified, labeled, and hybridized to Affymetrix Mouse Gene 1.0 ST Arrays.

ORGANISM(S): Mus musculus

SUBMITTER: CBDM Lab Diane Mathis Noriyuki Fujikado Christophe Benoist

PROVIDER: E-GEOD-66402 | ArrayExpress | 2015-03-07

SECONDARY ACCESSION(S): GSE66402PRJNA276790

REPOSITORIES: GEO, ArrayExpress

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Project description:The aim of this study is to evaluate the effect of Autoimmune regulator (Aire) gene disruption in a murine medullary thymic epithelial cells (mTEC 3.10 cell line) on the transcriptome of these cells during its adhesion with thymocytes. The mTEC-thymocyte adhesion is a crucial step for the negative selection of autoreactive thymocytes and prevention of autoimmune diseases. To generate Aire mutant cell clones, a total of 5x10^5 mTEC 3.10 cells were electro-transfected (Lonza Nucleofector) with CRISPR-Cas9 plasmid targeting the Aire Exon 3 (plasmid "all in one" encoding Aire Exon 3 gRNA + Cas9 + GFP, from Sigma-Aldrich). The GFP positive mTEC single cells were sorted by using a FACS Aria III cytometer and cells were cloned by expansion in culture. Sanger sequencing of PCR products from the Aire Exon 3 of these clones was used in order to evaluate the occurrence of indel mutations within the targeted Exon 3. The mTEC 3.10 clone E6 was identified and validated as a compound heterozygous Aire KO (Aire +/-). This clone features the Aire allele 1 that encodes a mutant Aire protein carring a neutral aminoacid substitution (A118P) and allele 2 encoding a truncated Aire protein. Wild type (WT) mTEC 3.10 cells or mTEC 3.10 clone E6 were cultured in the presence (or not) of thymocytes in order to establish cell adhesion. The total RNA preparations from WT or clone E6 mTEC cells (before or after mTEC- thymocyte co-cultures) were then sequenced through RNA-sequencing using a Illumina HiSeq 2500 instrument and the TruSeq Stranded mRNA Library Preparation kit resulting in about 50 million paired-end stranded specific 100 bp reads per sample. Sequencing reads were mapped to Mus musculus reference genome (mm10) using STAR v.2.5.0a. Read counts over transcripts were calculated using HTSeq v.0.6.1p2 based on a current UCSC annotation file for GRCm38/mm10 (Dec. 2011). Overall design: The mRNA profiles of mTEC 3.10 cells carring WT Aire (before or after co-culture with thymocytes) or heterozygous KO mTEC 3.10 cells (clone E6, Aire +/-) (before or after co-culture with thymocytes) were generated by sequencing, in duplicates, using a Illumina HiSeq 2500 instrument.