NextSeq 500 paired end sequencing; 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
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
Peripheral blood mononuclear cells (PBMCs) were isolated from sodium heparin anti-coagulated buffy coats using Lymphoprep (STEMCELL Technologies, Tullamarine, Australia) by density gradient centrifugation. The cells were washed once in sterile phosphate-buffered saline (PBS), and then in RPMI-1640 (Gibco BRL, Waltham, USA), before resuspension at 5-12×106/mL in complete medium comprised of RPMI-1640 supplemented with 20% (v/v) heat inactivated Fetal Bovine Serum (HI-FBS; Gibco BRL), 2 mM ʟ-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were cryopreserved in 90% complete medium/10% dimethyl-sulfoxide (DMSO) (v/v). Cryopreserved PBMCs were rapidly thawed in a 37°C water bath, 2.5×106 cells were washed twice with PBS, and stained with live dead stain and surface antibodies. A FACSAria™ III cell sorter (BD Biosciences) was used to sort different CD8 T cell subsets based on surface expression of CCR7, CD45RA, CD95, CD122, and CXCR3. TscmCD122+/- or CXCR3+/- were gated by CXCR3 or CD122 Fluorescence minus one (FMO) control. Cells were sorted based on the CD122 or CXCR3 fluorescence intensity. The TscmCD122hi population was defined as the upper tertile of CD122 expression and TscmCD122lo were defined as the lower tertile of expression of CD122. The TscmCXCR3hi and TscmCXCR3lo were defined similarly.  RNA-seq experiment was performed on mini-bulk sorted cells (n=50 cells per sample) to reduce heterogeneity and maintain an adequate transcriptomics signal. Adapted from Smartseq2 protocol from scRNAseq described by Picelli et al (Nat Proto, 2014), 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).