Single-Cell Transcriptome Analysis Of CD8+ T-cell Memory Inflation

We sought to define the nature of the transcriptional mechanisms underpinning memory inflation at different sites. We used single-cell RNA sequencing of M38-tetramer+-sorted cells from a single MCMV-infected mice, analyzing transcriptional networks in virus-specific populations in the spleen and gut intra-epithelial lymphocytes (IEL). Overall design: Illumina sequencing of single cells from murine M38 tetramer+ T cells from gut intraepithelium in comparison with murine M38 tetramer+ T cells from spleen. Lymphocytes were extracted from the spleen and gut intraepithelium by mechanical and enzymatic digestion. CD8+ T-cells were enriched by negative selection using the Miltenyi Biotec GmbH CD8a+ T-cell isolation kit (Bergisch Gladbach, Germany) . After M38-tetramer and surface staining, tetramer+ CD3+ CD8+ single-cells were sorted in two 96-well plates using a SH800S cell sorter (Sony, Tokyo, Japan). Sequencing libraries were prepared using the TruSeq dual-index sequencing primers (Illumina, San Diego, California) and paired-end sequencing was performed on the Illumina HiSeq4000 platform.