Computational protocol: Sex hormones have pervasive effects on thymic epithelial cells

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Protocol publication

[…] We analyzed the transcriptome of six populations of TECs: cTECs and mTECs from males, females and Cx males. We obtained three biological replicates from each TEC population, except for Cx male mTECs where we had 2 biological replicates. Hence, in toto, we analyzed 17 biological replicates (). Each biological replicate contained pooled FACs-sorted cTECs or mTECS from four to six mice. Total RNA was isolated using TrizolTM as recommended by the manufacturer (Invitrogen), and then further purified using RNeasy Micro columns (Qiagen). Sample quality was assessed using Bioanalyzer RNA Pico chips (Agilent). Transcriptome libraries were made using the TruSeq RNA Sample Prep Kit (v2) (Illumina) following the manufacturer’s protocols. Library generation was then assessed using a Bioanalyzer platform (Agilent) and Illumina MiSeq-QC run. Then, sequencing was done using an Illumina HiSeq2000 using TruSeq SBS v3 chemistry at the Institute for Research in Immunology and Cancer’s Genomics Platform. Cluster density was targeted at around 800 k clusters/mm2. Data was mapped to the Mus musculus (mm10) reference genome using the ELANDv2 alignment tool from the CASAVA 1.8.2 software (Illumina). RNA-seq data have been deposited in GEO archives (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE66873 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE66873) and are displayed in . Analyses of RNA sequencing data were performed using the publicly available statistical software package “R” (http://www.r-project.org/). Differentially expressed genes (DEGs) were determined with the DESeq package from Bioconductor (http://bioconductor.org/), using thresholds of adjusted p-value of 0.1 and a minimal fold change of 1.5. To remove genes that were lowly expressed in our analysis, we further filtered the DEGs to only keep genes that had at least one sample with a relative expression higher than 1 read per kilobase of exon model per million reads mapped (RPKM). Enrichment of biological functions and predicted upstream regulators were assessed using the IPA software (Ingenuity Systems, http://www.ingenuity.com). […]

Pipeline specifications

Software tools BaseSpace, DESeq, IPA
Application RNA-seq analysis
Organisms Mus musculus
Diseases Autoimmune Diseases