DOI: 10.5061/dryad.08kprr5cq
title: Data from: Single cell RNA-seq data of E18 fetal thymocytes from HEB Vav-iCre and Id3-KO mice and their wild type littermate counterparts

description: 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.
description: 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. 
description: # Data from: Single cell RNA-seq data of E18 fetal thymocytes from HEB
 Vav-iCre and Id3-KO mice and their wild type littermate counterparts
 Dataset DOI:
 [10.5061/dryad.08kprr5cq](https://doi.org/10.5061/dryad.08kprr5cq) ##
 Description of the data and file structure Title of dataset: scRNA-seq
 analysis of E18 fetal thymocytes lacking HEB or Id3 transcriptional
 regulators  Author/Principal Investigator Information Name: Michele K.
 Anderson ORCID: 0000-0002-8820-5910 Institution: Sunnybrook Research
 Institute, University of Toronto Address: 2075 Bayview Ave, M7-615,
 Toronto, ON Canada M4N 3M5
 Email: [manderso@sri.utoronto.ca](mailto:manderso@sri.utoronto.ca)   Date
 of data collection: December 2020 (HEB cKO), June 2022 (Id3-KO) Geographic
 location of data collection: Toronto, Canada Funding Sources: Canadian
 Institutes for Health Research, National Institutes of Health ###
 Description of the Project  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 the transcriptional regulators HEB and Id3 regulate γδT17
 cell development, we conducted single cell RNA-sequencing on 1) fetal
 thymocytes from mice with a germline deletion of Id3 in parallel with
 their WT littermates, and 2) fetal thymic γδ T cells from mice lacking HEB
 in all hematopoietic cells in parallel with their WT littermates. To
 obtain WT and Id3-KO cells, timed matings were performed by placing male
 and female Id3+/- mice together overnight. The pair was separated the next
 day to give a known day of conception for those mice that were pregnant.
 Embryos were harvested eighteen days later, and tail tissue was taken used
 for genotyping by PCR. HEB cKO embryos were obtained from timed matings of
 HEB cKO Vav-Cre+ and HEB cKO Vav-Cre- mice. Thymic lobes were harvested,
 and single cell suspensions were obtained by pushing the cells through
 wire mesh into ice cold 1XHBSS buffer. For HEB cKO experiments, cells of
 like genotypes were pooled and stained with antibodies that detect CD3 and
 TCRγδ, followed by isolation of CD3+γδTCR+ cells by fluorescence activated
 cells sorting.  For Id3-KO experiments,  cells of the same genotype were
 pooled and stained with antibody-labeled magnetic beads against CD4 and
 CD8. Double negative (DN; CD4-CD8-) cells, which are enriched for gd T
 cells, were isolated by magnetic sorting using MACS columns (Miltenyi). 
 HEB cKO gd T cells and Id3 DN cells were subjected to single cell RNA
 sequencing using the 10X Genomics platform, and data was analyzed using
 R-Seurat. ### Description of scRNA-sequencing technology and processing
 platform The HEB cKO dataset was generated with the 10X Genomics platform
 using the Chromium Next GEM Single Cell 5' Kit v2 (Dual Index). Next
 generation Illumina sequencing was performed to an approximate depth of
 25,000 reads. The count matrix was generated from raw FASTQ files.  The
 Id3-KO dataset was generated using the 10X Genomics platform using the
 Chromium Next GEM Single Cell 3' Kit v2 (Dual Index) (10X Genomics).
 Next generation Illumina sequencing was performed to an approximate depth
 of 100,000 reads. The count matrix was generated from raw FASTQ files. ###
 Files and variables Description of Samples: HEB cKO samples:
 Species: Mouse Strain: C57Bl/6 Allele 1 (HEB fl/fl): HEB flox/flox (normal
 WT), loxP sites surrounding the helix-loop-helix domain in the *Tcf12*gene
 locus abbreviated as HEB fl/fl Allele 2: Vav-Cre, deletes regions between
 loxP sites in all hematopoietic cells, including hematopoietic stem cells
 Control mice (“WT”): HEB fl/fl, without Vav-Cre, HEB intact Experimental
 mice(“HEB cKO”): HEB fl/fl, with Vav-Cre, HEB deleted from all
 hematopoietic cells Cells: sorted gamma delta (gd) T cells (gdT) Tissue:
 fetal thymus, embryonic day 18   Id3-KO samples: Species: Mouse Strain:
 C57Bl6 Allele (Id3-KO): red fluorescence protein (RFP) was inserted into
 the Id3 gene locus to create an Id3 RFP knock-in knock-out in mice (KI/KO)
 with homozygous mutant alleles. We bred these mice to obtain them on the
 B6 background. Control mice (“WT”): no RFP KI/KO allele Experimental mice
 (“Id3-KO”): Homozygous Id3 RFP KI/KO alleles Cells: Magnetic bead enriched
 double negative (CD4-CD8-) thymocytes Tissue: fetal thymus, embryonic day
 18 
 
 #### #Description of the Files HEBcKO_E18gd_scRNAseq_matrix/
                   -gdT_WT_matrix/                                     -
 HEBWT_cell_identity_barcodes.tsv.gz                                     -
 HEBWT_gene_name_features.tsv.gz                                     -
 HEBWT_gene_expression_levels_matrix.mtx.gz                   -
 gdT_HEBcKO_matrix/                                     -
 HEBcKO_gene_expression_levels_matrix.mtx.gz
                                     - HEBcKO_cell_identity_barcodes.tsv.gz
                                     - HEBcKO_gene_name_features.tsv.gz  
 Id3KO_E18_DN_scRNAseq_matrix/                   -Id3KO_matrix/
                                    
 -Id3KO_gene_expression_levels_matrix.mtx.gz
                                     -Id3KO_gene_name_features.tsv.gz
                                     -Id3KO_cell_identity_barcodes.tsv.gz
                   -Id3WT_matrix/                                    
 -Id3WT_gene_expression_levels_matrix.mtx.gz
                                     -Id3WT_gene_name_features.tsv.gz
                                     -Id3WT_cell_identity_barcodes.tsv.gz
 Final_for_HEBcKO.Rmd is the R-markdown file for the Seurat analysis code
 used to generate HEBcKO scRNA-seq figures, and HEBcKO_rmd.html file is the
 html output of the analysis run with that code. Final_for_Id3KO.Rmd is the
 R-markdown file for the Seurat analysis code used to generate Id3-KO
 scRNA-seq figures, and Id3KO_rmd.html file is the html output of the
 analysis run with that code. 
 
   ### Description of the Matrix Files  This directory contains the gene
 expression count matrix and associated metadata files generated from
 single-cell RNA sequencing data.  The data is provided as a set of three
 files in the standard 10x Genomics output format (compressed Matrix Market
 format). \`matrix.mtx.gz`: The core count data as a sparse matrix.
 \`features.tsv.gz`: Corresponds to the rows of the matrix, containing gene
 identifiers and symbols. \`barcodes.tsv.gz`: Corresponds to the columns of
 the matrix, containing cell barcodes. ### Matrix Structure and Content
  Dimensions: The matrix is a *gene-by-cell* matrix, where rows represent
 individual genes and columns represent individual cells. Data Type: The
 entries are *raw unique molecular identifier (UMI) counts*. Matrix values:
 Each value represents the number of UMIs mapped to a specific gene in a
 given cell. ## Code/software For both the HEB cKO and Id3-KO datasets, the
 count matrix was generated from raw FASTQ files using the following Cell
 Ranger software and the Alignment/Quantification tool STAR. HEB cKO
 sequences were aligned to the Reference Genome: Mouse, mm10. Id3-KO
 sequences were aligned to the Reference Genome: Mouse, mm39. ### Usage
 Example  To load and begin working with this data in a common
 bioinformatics environment, you might use a tool like Seurat in R.  R
 (Seurat): Use the `Read10X()` function to load the data directory. ###
 Code and software  scRNA-seq matrix files were analyzed using programs in
 R-Seurat. All code and programs are included in R-markdown files (RMD and
 html output)