Whole transcriptome analysis for T cell receptor-affinity and IRF4-regulated clonal expansion of T cells.
Clonal population expansion of T cells during an immune response is dependent on the affinity of the T cell receptor (TCR) for its antigen [1]. However, there is little understanding of how this process is controlled transcriptionally. We found that the transcription factor IRF4 was induced in a manner dependent on TCR-affinity and was critical for the clonal expansion and maintenance of effector function of antigen-specific CD8(+) T cells. We performed a genome-wide expression profiling experiment using RNA sequencing technology (RNA-seq) to interrogate global expression changes when IRF4 was deleted in CD8(+) T cells activated with either a low or high affinity peptide ligand. This allowed us not only to determine IRF4-dependent transcriptional changes but also to identify transcripts dependent on TCR-affinity [2]. Here we describe in detail the analyses of the RNA-seq data, including quality control, read mapping, quantification, normalization and assessment of differential gene expression. The RNA-seq data can be accessed from Gene Expression Omnibus database (accession number GSE49929).
The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia ; The Department of Computing and Information Systems, University of Melbourne, Parkville, Australia.
Wei
W
Shi
The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia ; The Department of Medical Biology, University of Melbourne, Parkville, Australia.
Kevin
K
Man
The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia ; The Department of Mathematics and Statistics, University of Melbourne, Parkville, Australia.
Gordon K
GK
Smyth
The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia ; The Department of Medical Biology, University of Melbourne, Parkville, Australia.
Stephen L
SL
Nutt
The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia ; The Department of Medical Biology, University of Melbourne, Parkville, Australia.
Axel
A
Kallies
Clonal expansion; IRF4; RNA-sequencing analysis; T cells; TCR-affinity