Computational protocol: Detection of Quiescent Radioresistant Epithelial Progenitors in the Adult Thymus

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Protocol publication

[…] We analyzed the transcriptome of two populations of TECs: UEA1– LRCs (GFPhi) and UEA1– NonLRCs (GFP–) from females after a 6-week pulse period and a 16-week chase period. We obtained one biological replicates of each TEC population from a pool of 11 mice to get a minimum of 104 cells per sample. Total RNA was isolated using Trizol™ 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 Illumina HiSeq2000 and 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 were 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 under accession number GSE94642 and are displayed in Table in Supplementary Material. Analyses of RNA-sequencing data were performed using the publicly available statistical software package “R.” To remove genes that were lowly expressed in our analysis, we considered only genes that had a relative expression higher than 1 RPKM in at least one sample. Enrichment of biological functions were performed using the Gene Functional Annotation tool from DAVID bioinformatics resources [version 6.8 (, )], and reduction of redundancy through semantic similarity was performed using REViGO web-based tool for gene ontology (GO) analysis ().RNA-Seq data for Sca1+ mesenchymal cells and for UEA1+ TECs from female mice were extracted from Patenaude and Perreault () and Dumont-Lagacé et al. (), respectively. Data can be found under GEO accession numbers GSE60101 and GSE66873. […]

Pipeline specifications

Software tools BaseSpace, DAVID, REViGO
Application RNA-seq analysis
Organisms Mus musculus
Diseases Brain Injuries, Heart Arrest