ATAC-seq data from DN3 and DN4 cells from WT, HEBcKO, and Rag2-KO mice
Data files
Mar 09, 2026 version files 1.08 GB
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DN3_HEBcKO1_ATAC_mm10.bw
126.45 MB
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DN3_HEBcKO2_ATAC_mm10.bw
126.47 MB
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DN3_Rag2KO1_ATAC_mm10.bw
93.74 MB
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DN3_Rag2KO2_ATAC_mm10.bw
99.71 MB
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DN3_WT1_ATAC_mm10.bw
97.93 MB
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DN3_WT2_ATAC_mm10.bw
108.96 MB
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DN4_HEBcKO1_ATAC_mm10.bw
116.02 MB
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DN4_HEBcKO2_ATAC_mm10.bw
106.16 MB
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DN4_WT1_ATAC_mm10.bw
98.94 MB
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DN4_WT2_ATAC_mm10.bw
101.40 MB
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README.md
4.17 KB
Abstract
Gamma delta T cells that produce IL-17 (gdT17) play essential roles in barrier immunity and autoimmunity, but the gene networks that install their functions are not well understood. We have shown that the transcriptional regulator HEB is required for efficient upregulation of Id3 in response to TCR signaling in DN3 cells, and that Id3 is essential for maturation of gdT17 cells. To evaluate how the loss of the HEB affects chromatin status in response to TCR signaling, we compared thymocyte subsets from WT and adult HEB fl/fl Vav-iCre (HEB cKO) mice, which lack HEB in all hematopoietic cells, by ATAC-seq. We sorted DN3 and DN4 thymocytes from adult WT mice and HEB cKO mice. We also sorted DN3 cells from adult Rag2-/- mice, which cannot rearrange their TCR genes and thus are unable to develop beyond the DN3 stage.
Dataset DOI: 10.5061/dryad.4tmpg4fr5
Description of the data and file structure
These data revealed that E protein binding sites in the Id3 locus are accessible in DN3 cells from WT and Rag2-KO mice, and from DN4 cells in WT and HEB cKO mice. However, DN3 cells from HEB cKO mice have dampened accessibility, indicating a role for HEB in remodeling of the Id3 locus prior to TCR signaling.
Thymuses were dissected from adult Rag2KO WT and HEB cKO mice, pressed through mesh to generate single cell suspensions, and incubated with Fc block. WT and HEB cKO DN cells were enriched by magnetic sorting using anti-CD4 and anti-CD8 microbeads according to the manufacturer’s instructions (Miltenyi Biotech). Flow-through (CD4-CD8-) cells were stained and flow sorted into two populations: DN3 (CD4-CD8-CD44-CD25+) cells and DN4 (CD4-CD8-CD44-CD25-). DN3 cells were sorted from unfractionated Rag2-KO thymocytes, which do not develop past the DN3 stage. Duplicates were generated for each subset, with each replicate derived from three mice. The sorted cells were cryopreserved in aliquots of 100,000 cells each, which served as the input material for bulk ATAC-sequencing.
Files and variables
Files and variables
This directory contains bigWIG (.bw) files that allow visualization of peaks representing accessible sites by alignment with the mm39 genome. Each folder is named with the thymocyte subset (DN3, DN4) and genotype (Rag2KO, WT, HEBcKO), and contains two replicates (1 and 2).
#Experimental Variables
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 Tcf12gene 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
HEB cKO mice: HEB fl/fl, with Vav-Cre, HEB deleted from all hematopoietic cells
Rag2KO mice (“Rag2-/-”): Rag2 gene germline deleted mice, lack thymocytes beyond the DN3 stage due to a defect in TCR rearrangement and signaling
Cells: sorted DN3 and DN4 thymocytes
Tissue: adult thymus, 6-9 weeks old
File: DN3_Rag2KO2_ATAC_mm10.bw
Description: ATAC-seq BigWIG file for sorted Rag2-KO DN3 cells, replicate 2
File: DN3_Rag2KO1_ATAC_mm10.bw
Description: ATAC-seq BigWIG file for sorted Rag2-KO DN3 cells, replicate 1
File: DN3_HEBcKO2_ATAC_mm10.bw
Description: ATAC-seq BigWIG file for sorted HEB cKO DN3 cells, replicate 2
File: DN3_HEBcKO1_ATAC_mm10.bw
Description: ATAC-seq BigWIG file for sorted HEB cKO, DN3 cells, replicate 1
File: DN4_HEBcKO2_ATAC_mm10.bw
Description: ATAC-seq BigWIG file for sorted HEB cKO DN4 cells, replicate 2
File: DN4_HEBcKO1_ATAC_mm10.bw
Description: ATAC-seq BigWIG file for sorted HEB cKO DN4 cells, replicate 1
File: DN4_WT2_ATAC_mm10.bw
Description: ATAC-seq BigWIG file for sorted WT DN4 cells, replicate 2
File: DN4_WT1_ATAC_mm10.bw
Description: ATAC-seq BigWIG file for sorted WT DN4 cells, replicate 1
File: DN3_WT1_ATAC_mm10.bw
Description: ATAC-seq BigWIG file for sorted WT DN3 cells, replicate 1
File: DN3_WT2_ATAC_mm10.bw
Description: ATAC-seq BigWIG file for sorted WT DN3 cells, replicate 2
Code/software
Raw sequencing data (FASTQ files) were processed as follows. Bcl2fastq2 (v2.20) was used for processing of Illumina base-call data and demultiplexing, and bwa (v0.7.12) was used to align of reads to reference genome (mm10). Samtools (v0.1.19) was used to process BAM files. BEDtools (v2.25.0) was used to process BED files, and wigToBigWig (v4) was used for generation of bigWIG files. bigWIG files were aligned using the Integrative Genomics Viewer (IgV) software (v2.16.2).
To load and begin working with this data in a common bioinformatics environment, you might use a program like Integrated Genomics Viewer (IgV) or the USCS Genome Browser.
Thymuses were dissected from adult Rag2-/-, WT, and HEB cKO mice, pressed through mesh to generate single cell suspensions, and incubated with Fc block. WT and HEB cKO DN cells were enriched by magnetic sorting using anti-CD4 and anti-CD8 microbeads according tothe manufacturer’s instructions (Miltenyi Biotech). Flow-through (CD4-CD8-) cells were stained and flow sorted into two populations: DN3 (CD4-CD8-CD44-CD25+) cells and DN4 (CD4-CD8-CD44-CD25-). DN3 cells were sorted from unfractionated Rag2-/- thymocytes, which do not develop past the DN3 stage. Duplicates were generated for each subset, with each replicate derived from three mice. The sorted cells were cryopreserved in aliquots of 100,000 cells each, which served as the input material for bulk ATAC-sequencing.
ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) is a method for determining chromatin accessibility across the genome. It utilizes a hyperactive Tn5 transposase to insert sequencing adapters into open chromatin regions. High-throughput sequencing then yields reads that indicate these regions of increased accessibility.
Sorted cells were cryopreserved in 50% FBS/40% growth media/10% DMSO in aliquots of 100,000 cells, which were subjected to ATAC-sequencing as follows. Cells were membrane permeabilized, and a transposase loaded with sequencing adaptors was added, mediating insertion of adaptors at accessible genomic locations. Libraries were amplified and subjected to paired-end next-generation sequencing using the Illumina platform. ATAC-seq libraries were generated and sequenced at Active Motif (Carlsbad, California).
Raw sequencing data (FASTQ files) were processed as follows. Bcl2fastq2 (v2.20) was used for processing of Illumina base-call data and demultiplexing, and bwa (v0.7.12) was used to align reads to the reference genome (mm10). Samtools (v0.1.19) was used to process BAM files. BEDtools (v2.25.0) was used to process BED files, and wigToBigWig (v4) was used for the generation of bigWIG files. bigWIG files were aligned using the Integrative Genomics Viewer (IgV) software (v2.16.2).