Single cell gene expression profile of GVHD-maintaining CD4 T cells isolated from spleen, colon, skin, and small intestine. Single cell gene expression profile of GVHD-maintaining CD4 T cells isolated from spleen, colon, skin, and small intestine

We used single cell RNA sequencing (scRNAseq) to analyze the transcriptional profiles of GVHD maintaining T cells in various GVHD target tissues Overall design: TS1 Tg CD4 cells were isolated from spleen, colon, skin, and small intestines (lamina propria and intraepithelial lymphocytes) of GVHD mice 4 weeks post-transplantation using fluorescence-activated cell sorting. After sorting, cells from each tissue were labelled with a unique TotalSeq-A hashing antibody and analyzed using scRNAseq HTO ID for Replicate 1: ACCCACCAGTAAGAC: Spleen_1 GGTCGAGAGCATTCA: Skin_1 CTTGCCGCATGTCAT: Colon_1 AAAGCATTCTTCACG: IEL_1 CTTTGTCTTTGTGAG: LP_1 HTO ID for Replicate 2: TATGCTGCCACGGTA: Spleen_2 GAGTCTGCCAGTATC: Skin_2 TATAGAACGCCAGGC: Colon_2 TGCCTATGAAACAAG: IEL_2 CCGATTGTAACAGAC: LP_2

我们采用单细胞RNA测序（single cell RNA sequencing, scRNAseq）分析了维持移植物抗宿主病（graft-versus-host disease, GVHD）的T细胞在不同GVHD靶组织中的转录组特征。
整体实验设计：于移植后4周，从GVHD模型小鼠的脾脏、结肠、皮肤及小肠中分离TS1转基因CD4⁺ T细胞，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）获取目标细胞，其中小肠样本包含固有层（lamina propria, LP）与上皮内淋巴细胞（intraepithelial lymphocytes, IEL）。分选完成后，为各组织的细胞添加独特的TotalSeq-A 哈希抗体标签，随后通过scRNAseq进行测序分析。
重复1的HTO标签：
ACCCACCAGTAAGAC：Spleen_1（脾脏_1）
GGTCGAGAGCATTCA：Skin_1（皮肤_1）
CTTGCCGCATGTCAT：Colon_1（结肠_1）
AAAGCATTCTTCACG：IEL_1（上皮内淋巴细胞_1）
CTTTGTCTTTGTGAG：LP_1（固有层_1）
重复2的HTO标签：
TATGCTGCCACGGTA：Spleen_2（脾脏_2）
GAGTCTGCCAGTATC：Skin_2（皮肤_2）
TATAGAACGCCAGGC：Colon_2（结肠_2）
TGCCTATGAAACAAG：IEL_2（上皮内淋巴细胞_2）
CCGATTGTAACAGAC：LP_2（固有层_2）