RARa restrains CD8 T cell effector program [bulk RNA-seq]

The roles of RARa and BATF on CD8 T cell gene expression have been determined. Overall design: For bulk RNA sequencing, CD8 T cells were cultured on anti-CD3-coated (1µg/ml) 96-wells in RPMI medium supplemented with 10% charcoal-stripped FBS, anti-CD28 (2 ug/ml), and IL-2 (100 U/ml) for 20 h with or without RA (15 nM). RNA was isolated using the RNAeasy kit (Qiagen #74104) according to manufacturers' instructions and processed for poly-A-enrichment-based library preparation for paired-end 150 bp-sequencing (300 cycle) in a NovaSeq S4 Shared Flowcell at the University of Michigan Advanced Genomics core. Duplicated samples were prepared for each group. BCL Convert Conversion Software v4.0 (Illumina) was used to generate de-multiplexed Fastq files. In general, Snakemake was used to manage the bioinformatics workflow 66. The reads were trimmed, examined for quality of data, and screened for various types of contamination. Reads were mapped to the reference genome GRCm38 (ENSEMBL) and assigned count estimates to genes with RSEM v1.3.3 using STAR v2.7.8a 67. Hierarchical clusters were generated using the Gene Cluster software (3.0) with a cut-off of =1.5 log2 fold-change and data was presented using the Java TreeView software.