Single cell RNA-seq data of E18 fetal thymocytes from HEB Vav-iCre and Id3-KO mice and their wild type littermate counterparts

γδ T cells that produce IL-17 (γδT17) play essential roles in barrier immunity and autoimmunity, but the gene networks that install their functions are not well understood. To understand how HEB and Id3 regulate γδT17 cell development, we conducted single-cell RNA-sequencing on fetal thymic γδ T cells from Tcf12-deficient (HEB cKO) and Id3 knockout (Id3-KO) E18 embryos. Four datasets were generated. The first two consist of WT and HEB cKO datasets derived from sorted gamma-delta T cells. The second two consist of WT and Id3-KO datasets derived from magnetically enriched for CD4/CD8-negative cells. HEB datasets were generated with 10X Genomics 5' chemistry, and Id3 datasets were generated with 10X Genomics 3' chemistry. Raw sequence files were processed using Cell Ranger to generate matrix files, which have been uploaded here. We also include R-markdown files (RMD) and R-markdown HTML output files to provide code and programs used for the data analysis shown in the associated paper.

Methods
Timed matings were set up with HEBfl/fl Vav-iCre (HEB cKO) mice and HEB fl/fl (HEB WT) mice, and for Id3-KO and WT (Id3-WT) mice. Embryos were harvested 18 days later (E18). Fetal thymuses were dissected and genotyped, and cells were pooled into four groups by genotype. Single cell suspensions were made by pushing the cells through wire mesh into 1XHBSS. For HEB mice, cells were stained with antibodies for CD3 and TCRγδ, and CD3+γδTCR+ cells were sorted and subjected to scRNA-seq using the Chromium Next GEM Single Cell 5' Kit v2 (Dual Index) (10X Genomics). For Id3 mice, CD4-CD8- cells were enriched by magnetic sorting and subjected to scRNA-seq using the Chromium Next GEM Single Cell 3' Kit v2 (Dual Index) (10X Genomics). For HEB cKO samples, next-generation Illumina sequencing was performed to a depth of ~20,000 reads, whereas Id3-KO samples were sequenced to a depth of approximately 100,000 reads. HEB cKO fastq files were aligned to the mouse mm10 genome, and Id3-KO fastq files were aligned to the mouse mm39 genome. These alignments were used to generate matrix files, which can be analyzed using R-Seurat or other scRNA-seq analysis packages.