NextSeq 550 paired end sequencing; scRNA-seq of allo-reactive CD4+ T cells
scRNA-seq of allo-reactive CD4+ T cells
C57BL/6J and BALB/c mice were purchased from the Animal Resources Centre (Canning Vale, Australia), transgenic TEa (Vα2+, Vβ6+, CD45.1+, CD90.1+) mice were bred in-house (4, 5). All mice were female and aged between 6 – 12 weeks of age and were maintained under specific pathogen-free conditions within the animal facility at QIMR Berghofer Medical Research Institute. Bone marrow transplantation. Balb/c mice were transplanted as previously described (4). Briefly, BALB/c mice received 900 cGy total body irradiation (TBI; 137Cs source at 84 cGy/min) on day -13. On day -12, Balb/c mice were transplanted with 10 x 10^6 bone marrow cells and 2 x 10^5 FACs purified T cells from C57BL/6J donor mice. On day 0, recipient BALB/c mice were injected intravenously with 1-2 x 10^6 FACS sorted TEa T cells (Vβ6+Vα2+CD45.1+). Cell Trace™ CFSE Cell Proliferation (Life Technologies) and Violet Proliferation Dye 450 (VPD450; BD Biosciences) staining were performed according to manufacturer's protocol. Reference: Koyama M, et al. Donor colonic CD103+ dendritic cells determine the severity of acute graft-versus-host disease. J Exp Med. 2015;212(8):1303-21. Grubin CE, Kovats S, deRoos P, and Rudensky AY. Deficient positive selection of CD4 T cells in mice displaying altered repertoires of MHC class II-bound self-peptides. Immunity. 1997;7(2):197-208. TEa cells from the mesenteric lymph node, small intestine and colon were isolated by flow cytometry into a 1% BSA/PBS buffer. Cells were assessed for viability by staining with 7AAD (Sigma-Aldrich), according to manufacturer's protocol. Prior to staining, cells were incubated with antibodies against CD16 and CD32 (2.4G2) to block Fc receptors. For surface staining, cells were incubated with the following antibodies for 20 minutes at 4°C: Vα2 (B20.1) – APC Cy7, CD45.1 (A20) – PeCy7 (all Biolegend). TEa cells were FACs isolated as Vα2+CD45.1+ cells. Approximately 8,000 cells were loaded per channel onto a Chromium controller (10x Genomics) for generation of gel-bead-in-emulsions.  Sequencing libraries were prepared using Single Cell 3' Reagent Kits v2 (mGVHD1 and mGVHD2) or v3.0 (mGVHD3) (10x Genomics).
QIMR Berghofer Medical Research Institute, Herston, Brisbane, QLD 4006, Australia.
E-MTAB-9125:mGVHD3 D5.Gut (IEL)_p