Single cell RNA sequencing of mouse donor T cells after graft-versus-host disease (GVHD) model

Lethally irradiated mice were transferred with bone marrow cells together with allogeneic Treg cells isolated from CD226+/+ TIGIT+/+, CD226-/- TIGIT+/+, CD226+/+ TIGIT-/-, or CD226-/- TIGIT-/- mice, followed by allogeneic wild-type Tconv cells two days later to induce acute GVHD. We performed scRNA sequencing of donor T cells isolated from the spleen of recipient mice eight days after GVHD induction. Mice on the BALB/c background were transferred after lethal irradiation with T cell-depleted bone marrow cells derived from wild-type C57BL/6 mice together with Treg cells (CD4+ Foxp3-GFP+ CD25hi) isolated from the spleen of CD226+/+ TIGIT+/+, CD226-/- TIGIT+/+, CD226+/+ TIGIT-/-, or CD226-/- TIGIT-/- mice on the C57BL/6 background, followed by Treg cell-depleted Tconv cells derived from wild-type C57BL/6 mice two days later to induce acute GVHD. PI- H-2Kb+ TCRb+ donor T cells isolated from the spleen of recipient mice eight days after GVHD induction. Three donor T cells were pooled per each sample. To distinguish between Tconv and Treg cells, the sorted donor T cells were re-stained with barcode sequence-conjugated anti-mouse CD45.1 and CD45.2 antibodies. The viability of all samples were more than 85%. Single cells were processed through the Chromium Single Cell Platform using the Chromium Single Cell 3’ Library and Gel Bead Kit v3 (10X Genomics, PN-1000092), the Chromium Single Cell B Chip Kit (10X Genomics, PN-1000074), and the TotalSeq™-A Antibodies with 10x Single Cell 3' Reagent Kit v3 (BioLegend) as per the manufacturer’s protocol. The sequencing of libraries was performed at Macrogen Japan (Kyoto, Japan) using an Illumina HiSeq X. Processed files were generated from Cell Ranger (10X Genomics) pipeline.