ATAC-seq data from DN3 and DN4 cells from WT, HEBcKO, and Rag2-KO mice

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.

Methods
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).

分泌白细胞介素17（IL-17）的γδ T细胞（Gamma delta T cells，简称gdT17）在屏障免疫与自身免疫过程中发挥关键作用，但介导其功能建立的基因调控网络仍未被充分阐明。本团队此前研究证实，转录调节因子HEB可在DN3细胞中响应T细胞受体（TCR）信号并有效上调分化抑制因子3（Id3）的表达，且Id3对于gdT17细胞的成熟至关重要。为探究HEB缺失如何影响TCR信号响应过程中的染色质状态，我们通过ATAC-seq（转座酶可及性高通量测序，Assay for Transposase-Accessible Chromatin with high-throughput sequencing）比较了野生型（wild type, WT）与成年HEB fl/fl Vav-iCre（HEB条件性敲除，HEB cKO）小鼠的胸腺细胞亚群——该HEB cKO小鼠在所有造血细胞中均缺失HEB。我们分别从成年WT小鼠与HEB cKO小鼠中分选了DN3与DN4胸腺细胞；同时从成年重组激活基因2敲除（Rag2-/-）小鼠中分选DN3细胞，此类小鼠无法完成TCR基因重排，因此发育停滞于DN3阶段。

实验方法
我们从成年Rag2-/-、WT及HEB cKO小鼠中分离胸腺，通过滤网研磨制备单细胞悬液，并使用Fc封闭试剂进行孵育。按照Miltenyi Biotech厂商说明书，我们利用抗CD4与抗CD8磁珠通过磁分选富集WT与HEB cKO小鼠的DN细胞。收集流出的CD4⁻CD8⁻细胞进行染色，通过流式分选得到两个细胞群：DN3细胞（CD4⁻CD8⁻CD44⁻CD25⁺）与DN4细胞（CD4⁻CD8⁻CD44⁻CD25⁻）。同时我们从未经分馏的Rag2-/-胸腺细胞中分选DN3细胞，此类小鼠无法完成DN3阶段之后的发育。每个细胞亚群均设置生物学重复，每一份重复样本来自3只小鼠。将分选得到的细胞以每管10万个细胞的量冻存，作为批量ATAC测序的起始材料。

ATAC-seq（转座酶可及性高通量测序，Assay for Transposase-Accessible Chromatin with high-throughput sequencing）是一种用于全基因组染色质可及性检测的技术。该方法利用高活性Tn5转座酶将测序接头插入开放染色质区域，随后通过高通量测序获得可反映染色质高可及性区域的读段。

分选得到的细胞以50%胎牛血清（FBS, Fetal Bovine Serum）/40%培养液/10%二甲基亚砜（DMSO, Dimethyl Sulfoxide）的体系冻存，每管10万个细胞，随后按照下述流程进行ATAC测序：首先对细胞进行膜通透处理，加入加载有测序接头的转座酶，使其在基因组可及区域介导接头插入；随后扩增构建测序文库，并使用Illumina平台进行双端下一代测序。本研究的ATAC-seq文库构建与测序工作均在加利福尼亚州卡尔斯巴德的Active Motif公司完成。

原始测序数据（FASTQ格式文件）的处理流程如下：使用Bcl2fastq2（v2.20）处理Illumina碱基识别数据并完成解复用；使用bwa（v0.7.12）将测序读段比对至参考基因组mm10；使用Samtools（v0.1.19）处理BAM格式文件；使用BEDtools（v2.25.0）处理BED格式文件；使用wigToBigWig（v4）生成bigWIG格式文件。最终使用整合基因组浏览器（Integrative Genomics Viewer, IGV，v2.16.2）对bigWIG文件进行可视化分析。