RNA-seq from sorted population of CD8 T cells (50 cells per sample) of human CD8 T memory stem cells (Tscm) subsets defined by surface expression of CD122 or CXCR3

ABSTRACT: Although it is recognised that CD8+ T memory stem cells (Tscm) are heterogeneous in phenotype and function, very few studies have sought to systematically examine these characteristics, particularly in human samples, as the population as a whole is rare and antigen-specific cells even more so. For each T cell subsets investigated, we have performed optimized RNAseq for small number of cells by adapting the Smartseq2 protocol from scRNAseq. The mini-bulk number of cells was chosen to reduce heterogeneity but also maintain an adequate transcriptomics signal. Adapted from Smartseq2 protocol from scRNAseq described by Picelli et al, cell lysis buffer was prepared by adding 1 μl RNase inhibitor (Clontech, Mountain View, USA) to 19 μl Triton X‐100 solution (0.2% v/v). CD8 T cell subpopulations (TN, TSCM, TCM and TEM together with TSCM CXCR3hi/lo and CD122hi/lo) were bulk sorted (50 cells/sample, with replicates) directly into 96-well PCR plates containing 2 μl lysis buffer, 1 μl dNTP mix (10 mM) and 1 μl oligo‐dT primer at 5 μM. Plates were stored at -80 °C until ready to perform the assay. Reverse-transcription and PCR amplifications were performed as described, with the exception of reducing the IS PCR primer to a 50 nM final concentration and increasing the number of cycles to 28. Sequencing libraries were prepared using the Nextera XT Library Preparation Kit (Illumina; San Diego, USA) and sequencing was performed on Illumina NextSeq500 sequencing platforms with 2×150bp paired-end read output, the read depth was approximately 1.3 million reads per sample.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Homo sapiens

SUBMITTER: Yanran Zhao 

PROVIDER: E-MTAB-10208 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress