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ATAC seq of mouse CD4 T cells

ABSTRACT: Purpose: To gain more mechanistic insights into the effects of SDHB deletion on T cells, we performed ATAC seq in activated WT and SDHB cKO T cells. Methods: 5x104 cells per experiment were first washed with RSB buffer and gently permeabilized with RSB lysis buffer on ice. Cells were suspended in 50 uL of tagmentation master mix prepared from Illumina Tagment DNA TDE1 Enzyme and Buffer Kit components (#20034197), and transposition was performed for 30 minutes at 37°C. Tagmented DNA fragments were isolated using Qiagen MinElute PCR Purification columns prior to library amplification. ATAC-seq libraries were amplified with barcoded Nextera primers for 14 cycles, and excess primers were removed by size selection with AMPure XP beads. Libraries were sequenced on the HiSeq4000 platform running in PEx150bp mode. Results: ATAC-seq revealed distinctive patterns of DNA accessibility that are associated with genomic loci of genes involved in T cell activation and inflammation in activated WT and SDHB cKO CD4 T cells Conclusions: DNA accessibility that is associated with genomic loci of inflammatory genes and transcription factors that regulate inflammatory genes in CD4 T cells

ORGANISM(S): Mus musculus

PROVIDER: GSE184742 | GEO | 2022/03/08

REPOSITORIES: GEO

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Project description:Purpose: The goals of this study are to compare the pDC chromatin structure (ATAC-seq) at steady state and at 2h after CpG activation. Methods: Cells were harvested and frozen in culture media containing FBS and 5% DMSO. Cryopreserved cells were sent to Active Motif to perform the ATAC-seq assay. The cells were then thawed in a 37°C water bath, pelleted, washed with cold PBS, and tagmented as previously described (Buenrostro et al., 2013), with some modifications based on (Corces et al., 2017). Briefly, cell pellets were resuspended in lysis buffer, pelleted, and tagmented using the enzyme and buffer provided in the Nextera Library Prep Kit (Illumina). Tagmented DNA was then purified using the MinElute PCR purification kit (Qiagen), amplified with 10 cycles of PCR, and purified using Agencourt AMPure SPRI beads (Beckman Coulter). Resulting material was quantified using the KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems), and sequenced with PE42 sequencing on the NextSeq 500 sequencer (Illumina). Results: Reads were aligned using the BWA algorithm (mem mode; default settings). Duplicate reads were removed, only reads mapping as matched pairs and only uniquely mapped reads (mapping quality >= 1) were used for further analysis. Alignments were extended in silico at their 3’-ends to a length of 200 bp and assigned to 32-nt bins along the genome. The resulting histograms (genomic “signal maps”) were stored in bigWig files. Peaks were identified using the MACS 2.1.0 algorithm at a cut off of p-value 1e-7, without control file, and with the –nomodel option. Peaks that were on the ENCODE blacklist of known false ChIP-Seq peaks were removed. Signal maps and peak locations were used as input data to Active Motifs proprietary analysis program, which creates Excel tables containing detailed information on sample comparison, peak metrics, peak locations and gene annotations. For differential analysis, reads were counted in all merged peak regions (using Subread), and the replicates for each condition were compared using DESeq2. Conclusions: Our study represents a detailed analysis of naive and activated pDC chromatin that analyzes the role of the transcription factor BATF for chromatin structure in pDCs.

Project description:Non-coding regions of the genome contain cis-regulatory elements (CREs) that govern transcriptional activity critical for development and physiologic functions of major metabolic tissues, such as islets, adipose, liver, and skeletal muscle. Moreover, they contain the majority of genetic variants associated with risk of type 2 diabetes and quantitative alterations in related metabolic phenotypes. Here, we defined comprehensive, representative CRE maps of these major metabolic tissues in mice of both sexes using ATAC-seq and incorporating genetic variation of the 8 major Collaborative Cross founders and diet-response. Cross-species comparison of these representative maps with those from corresponding human tissues revealed conservation and functional preservation of ~28% of CREs in one or more major metabolic tissues. Importantly, this included an average of 700 (1.8%) cross-species concordant peaks that harbored genetic variants associated with T2D and related metabolic traits, including variants in the CAMK1D/CDC123, C2CD4A/B, and ADCY5 loci. Germline knock-out of the cross-species concordant CRE in ADCY5 yielded mice with impaired fasting glucose, demonstrating the importance of these functionally preserved CREs to glucose homeostasis and genetic risk of type 2 diabetes and progression. Together, the creation and cross-species comparison of comprehensive CRE maps in major metabolic tissues provides a critical resource for variant-to-function studies of type 2 diabetes and an array of related metabolic and cardiovascular traits and demonstrates the power of these mapping strategies to identify conserved metabolic cis-regulatory networks and enable data-driven creation of new preclinical models of diabetes and related metabolic diseases. Sample preparation: ATAC-seq libraries were prepared from adipose, liver, muscle, and islets as described in [Corces et al., 2016 (Nature Genetics) with slight modifications. Adipose, liver, and muscle samples were dissociated into single cell suspensions using MACS Miltenyi Biotec Adipose Tissue (130-105-808), Liver (130-105-807), and Skeletal Muscle (130-098-305) Dissociation Kits. Dissociated cells were counted and 50,000 cells were resuspended in freezing media (10% DMSO, 50% FBS, 40% RPMI) and frozen at -80°C until cells were thawed and washed in cold PBS, and incubated in ATAC lysis buffer. Islets were isolated from total pancreas as described in [Simonett et al., 2021 (JCI)], and 50 islets were rested overnight in RPMI in 37°C incubator. The following day islets were washed in cold PBS, incubated in ATAC lysis buffer, and triturated with 25-gauge needle and centrifuged to generate a crude nuclei pellet, and washed in ATAC lysis buffer. Prepared nuclei from all tissues were transposed using the Tagment DNA TDE1 Enzyme and Buffer kit (Illumina) with the addition of 0.01% digitonin (1x TDE for liver, muscle; 2x TDE for adipose). Transposed DNA was purified using the ChIP DNA Clean & Concentrator Kit (Zymo Research), amplified with 11 cycles of PCR, and cleaned using KAPA Pure Beads (Roche). All ATAC-seq libraries were sequenced at 2 x 100 bp using Illumina NovaSeq 6000. 1. Corces M.R., Buenrostro J.D., Wu B., Greenside P.G., Chan S.M., Koenig J.L., Snyder M.P., Pritchard J.K., Kundaje A., Greenleaf W.J. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nat. Genet. 2016;48:1193–1203. https://www.nature.com/articles/ng.3646 2. Simonett S.P., Shin S., Herring J.A., Bacher R., Smith L.A., Dong C., Rabaglia M.E., Stapleton D.S., Schueler K.L., Choi J., Bernstein M.N., Turkewitz D.R., Perez-Cervantes C., Spaeth J., Stein R., Tessem J.S., Kendziorski C., Keleş S., Moskowitz I.P., Keller M.P., Attie A.D. Identification of direct transcriptional targets of NFATC2 that promote beta-cell proliferation. J Clin Invest. 2021;131(21):e144833. https://doi.org/10.1172/JCI144833.

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ATAC seq of mouse CD4 T cells

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