BioProject

Total RNA of regulatory T (Treg) cells and conventional CD4 T (Tconv) cells from young WT (2 months), aged WT (>18 months) and young Dcaf1-deficient mice was extracted and RNA-seq libraries were generated. More...

Total RNA of regulatory T (Treg) cells and conventional CD4 T (Tconv) cells from young WT (2 months), aged WT (>18 months) and young Dcaf1-deficient mice was extracted and RNA-seq libraries were generated. Reads (32-45 Million reads per sample) were analyzed with Salmon software to align and quantify the transcript expression. R packages in Bioconductor, tximport and tximportData were used to aggregate transcript-level quantifications to the gene level, with the R package biomaRt for gene and transcripts mapping. The option "lengthScaledTPM" for countsFromAbundance in tximport was used to obtain the estimated counts at the gene level using abundance estimates scaled based on the average transcript length over samples and the library size. The option "lengthScaledTPM" for countsFromAbundance in tximport was used to obtain the estimated counts at the gene level using abundance estimates scaled based on the average transcript length over samples and the library size. For the differential expression (DE) analysis of RNA-seq data, gene-level count matrix was passed into by DESeq2 package as input directly from the tximport package. The normalized gene expression data was retrieved from DESeq2 analysis after regulated log (rlog) transformation (‘rlog’ in DESeq2). The z-score at gene-level average of normalized expression matrix was used to generate heatmap in Gene-E from Broad Institute (www.broadinstitute.org/ /GENE-E/). Gene Set Enrichment Analysis was performed using the Java application available from Broad Institute (www.broadinstitute.org/gsea/). Gene set databases including Hallmarks (h.all.v6.1.symbols.gmt) and KEGG (c2.cp.kegg.v6.1.symbols.gmt) from the Molecular Signatures Database (MSigDB) were used in the analysis. The aging-program gene set was from DEMAGALHAES_AGING_UP in MSigDB. One thousand gene set permutations were performed. FDR<0.05 was used for enriched terms, as is recommended when performing permutations by gene set. Overall design: Examination of differential gene expression in regulatory T (Treg) cells and conventional CD4 T (Tconv) cells from young WT (2 months), old WT (>18 months) and young (2 months) Dcaf1-deficient mice. Less...