Single-cell RNAseq (10x Genomics) analysis of mouse splenic CD4+ T cells in WT and deltaFoxp3 mice and in WT/deltaFoxp3 bone marrow chimeras

Single-cell RNAseq (10x Genomics) analysis of mouse splenic CD4+ T cells in WT and deltaFoxp3 mice and in WT/deltaFoxp3 bone marrow chimeras. Mouse CD4+T cells in 21-day-old male WT and deltaFoxp3 mice were isolated from spleen by flow cytometry as DAPI-TCRbeta+CD4+ cells for 10x Genomics Single Cell 3' Reagent Kit (V2 chemistry, one sample per channel). For bone marrow chimera experiment: 7 week-old CD45.2-recipient mice were irradiated with 1000 Rad, reconstituted with 4 million CD3-depleted bone marrow cells: 50% CD45.1 x Foxp3-IRES-GFP (WT, 21d-old male) and 50% Foxp3DeltaEGFPiCre/RFP x ROSA-YFP x CD45.1/2 (scurfy, 21d-old male). 10 weeks later, spleen were harvested and tagged using a different Hashtags fo each mouse. deltaFoxp3 CD4+ T cells were sorted as DAPI-TCRb+CD4+CD45.1+CD45.2+. WT CD4+ T cells were sorted as DAPI-TCRb+CD4+CD45.1+CD45.2-. Control WT DAP-TCRb+CD4+GFP+ Treg cells and GFP- Tconvs cells were also tagged and sorted. Samples with different hastags were pooled before single cell encapsulation using 10x Genomics Single Cell 3' Reagent Kit (V3 chemistry). Mouse CD4+T cells in 21-day-old male WT and deltaFoxp3 mice were isolated from spleen by flow cytometry as DAPI-TCRbeta+CD4+ cells for 10x Genomics Single Cell 3' Reagent Kit (V2 chemistry, one sample per channel). For bone marrow chimera experiment: 7 week-old CD45.2-recipient mice were irradiated with 1000 Rad, reconstituted with 4 million CD3-depleted bone marrow cells: 50% CD45.1 x Foxp3-IRES-GFP (WT, 21d-old male) and 50% Foxp3DeltaEGFPiCre/RFP x ROSA-YFP x CD45.1/2 (scurfy, 21d-old male). 10 weeks later, spleen were harvested and tagged using a different Hashtags fo each mouse. deltaFoxp3 CD4+ T cells were sorted as DAPI-TCRb+CD4+CD45.1+CD45.2+. WT CD4+ T cells were sorted as DAPI-TCRb+CD4+CD45.1+CD45.2-. Control WT DAPI-TCRb+CD4+GFP+ Treg cells and GFP- Tconvs cells were also tagged and sorted. Samples with different hastags were pooled before single cell encapsulation using 10x Genomics Single Cell 3' Reagent Kit (V3 chemistry).本数据集针对野生型（Wild Type，WT）与deltaFoxp3小鼠，以及WT/deltaFoxp3骨髓嵌合体小鼠的脾脏CD4+ T细胞开展单细胞RNA测序（single-cell RNAseq）分析，所用平台为10x Genomics。 针对21日龄雄性WT及deltaFoxp3小鼠，通过流式细胞术以DAPI-TCRβ+CD4+为表型从脾脏中分选CD4+ T细胞，使用10x Genomics单细胞3'端转录组试剂盒（V2化学试剂，每个通道对应一个样本）开展实验。 骨髓嵌合体实验构建方案如下：选取7周龄CD45.2受体小鼠，以1000拉德（Rad）剂量进行辐照，随后移植4×10^6个经CD3耗竭处理的骨髓细胞，细胞配比为50% CD45.1×Foxp3-IRES-GFP（WT，21日龄雄性）与50% Foxp3DeltaEGFPiCre/RFP×ROSA-YFP×CD45.1/2（scurfy突变小鼠，21日龄雄性）。移植10周后收获小鼠脾脏，为每只小鼠分配不同的哈希标签（Hashtags）进行标记。其中deltaFoxp3 CD4+ T细胞的分选表型为DAPI-TCRβ+CD4+CD45.1+CD45.2+；WT CD4+ T细胞的分选表型为DAPI-TCRβ+CD4+CD45.1+CD45.2-。同时同步标记并分选野生型对照的DAPI-TCRβ+CD4+GFP+调节性T细胞（regulatory T cells，Treg）与GFP-常规T细胞（Tconvs）。将带有不同哈希标签的样本混合后，使用10x Genomics单细胞3'端转录组试剂盒（V3化学试剂）进行单细胞包裹操作。 针对21日龄雄性WT及deltaFoxp3小鼠，通过流式细胞术以DAPI-TCRβ+CD4+为表型从脾脏中分选CD4+ T细胞，使用10x Genomics单细胞3'端转录组试剂盒（V2化学试剂，每个通道对应一个样本）开展实验。 骨髓嵌合体实验构建方案如下：选取7周龄CD45.2受体小鼠，以1000拉德（Rad）剂量进行辐照，随后移植4×10^6个经CD3耗竭处理的骨髓细胞，细胞配比为50% CD45.1×Foxp3-IRES-GFP（WT，21日龄雄性）与50% Foxp3DeltaEGFPiCre/RFP×ROSA-YFP×CD45.1/2（scurfy突变小鼠，21日龄雄性）。移植10周后收获小鼠脾脏，为每只小鼠分配不同的哈希标签（Hashtags）进行标记。其中deltaFoxp3 CD4+ T细胞的分选表型为DAPI-TCRβ+CD4+CD45.1+CD45.2+；WT CD4+ T细胞的分选表型为DAPI-TCRβ+CD4+CD45.1+CD45.2-。同时同步标记并分选野生型对照的DAPI-TCRβ+CD4+GFP+调节性T细胞（regulatory T cells，Treg）与GFP-常规T细胞（Tconvs）。将带有不同哈希标签的样本混合后，使用10x Genomics单细胞3'端转录组试剂盒（V3化学试剂）进行单细胞包裹操作。