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| Status |
Public on Feb 24, 2024 |
| Title |
ATAC_T8_STAT1KO_H_H_BSMJ002 |
| Sample type |
SRA |
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| Source name |
Murine spleenic cells
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| Organism |
Mus musculus |
| Characteristics |
organ: spleen
cell type: CD8 T cell
genotype: Stat1-ko
treatment: Homeostatic
age: 8-12 weeks
strain: C57BL/6
research group: BS
batch: MJ002
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| Treatment protocol |
In vitro culture and treatment of ex vivo sort purified cells was done following this protocol: Aliquots of the sort-purified cell populations were stored for RNA/DNA isolation in RLT-buffer (Qi-agen) or directly processed using the ATAC-seq assay (as described below). Splenic macrophages and CD8+ T cells were cultured in 48 well tissue culture treated plates for in vitro treatment with different stimuli. To that end, the cells were centrifuged at 500g for 5 min at 4 °C. 1*105 macrophages and 3*105 T cells were seeded per well in 300µl media. Macrophages were resuspended in DMEM (10% FCS, 5 ml penicillin streptomycin with 10,000 U/ml) and T cells in RPMI (10% FCS, 5 mL penicillin streptomycin with 10000 U/ml). The following conditions were applied: (i) 20 hours in culture untreated, (ii) 20 hours in culture with treatment, and (iii) 18.5 hours in culture followed by treatment for the last 1.5 hours. Treatments included murine recombinant IFN-β carrier-free (PBL as-say science, Cat. No. 12401-1) at a final concentration of 1,000 U/ml or recombinant murine IL-2 (PeproTech, Cat. No. 212-12) at a final concentration of 1,000 ng/ml or murine M-CSF (PeproTech, Cat. No. 315-02) at a final concentration of 100 ng/ml. T cells were harvested by transferring them into a reaction tube, additional cold PBS (0.2% BSA) was added, centrifuged at 500g for 5 min at 4 °C and supernatant was removed. T cells were resuspended in 1ml PBS (0.2% BSA) and split equally between the two tubes. Macrophages were harvested by remov-ing the supernatant, gently rinsed the cells with cold PBS (0.2% BSA), followed by the addition of cold PBS (0.2% BSA). Macrophages were scraped and equally split between the two tubes. Tubes were then centrifuged at 500g for 5 min at 4 °C and either taken for RNA/DNA isolation or ATAC-seq (as de-scribed below). After centrifugation, the supernatant was carefully removed and the pellet was resus-pended in 350μl RLT buffer (Qiagen) with 3.5µl ß-mercaptoethanol (Sigma). After vortexing the sam-ple for one minute, it was stored at -80°C until further processing. RNA and DNA were isolated with the AllPrep RNA/DNA Micro Kit (Qiagen) following the manufacturer’s instructions and stored as recommended.
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| Growth protocol |
Ex vivo harvesting of cells was conducted following this protocol: We established and validated a robust isolation and staining protocol, which was used consistently across all experiments. Spleens were resected and immediately placed into tubes containing cold PBS (Gibco). Tissue was smashed with a 100µm strainer (SPL Life Sciences) using a syringe plunger and a 50ml tube. A new strainer was used for each spleen and rinsed with 10 to 20ml DMEM (Gibco) con-taining 10% FCS (Sigma) and 5ml penicillin streptomycin with 10,000 U/ml (Gibco). For the isolation of dendritic cells (DCs) spleens were injected with and placed in a digestion mixture (RPMI (Sigma), 2% FBS, 1mg/ml Collagenase D, 20µg/ml DNase I) and then incubated at 37°C for 30 minutes in a 24-well cell culture dish, before proceeding with the same mashing through a 100µm strainer. We pooled three littermates to obtain sufficient cell numbers. Samples were centrifuged at 500g for 5 min at 4 °C. Pellets were resuspended in 1ml Red Blood Cell Lysis Solution (Promega, Z3141) and incubated for 5min on ice. The lysis was stopped by adding 50ml 1x PBS. Samples were centrifuged at 500g for 5 min at 4 °C. Supernatant was discarded and pellets were resuspended in 1ml PBS supplemented with 2% BSA (Sigma). Samples were filtered through a 70µm strainer (SPL Life Sciences). The strainer was washed with 1ml PBS supplemented with 2% BSA. MHCII+ CD11c+ DCs were isolated from a DC MACS enriched population. Dendritic cells were enriched using the Miltenyi Pan Dendritic Cell Isola-tion Kit (mouse) according to the manufacturer’s instructions (Miltenyi Biotec, 130-100-875). Samples were centrifuged at 500g for 5 min at 4 °C and supernatant was discarded. Cell pellets were resuspended in 100µl PBS (2% BSA) and anti-CD16/CD32 (clone 93, Biolegend) was added at a concentration of 1:500 for 15 min to prevent nonspecific binding. Single-cell suspensions were then stained with combinations of antibodies against TER-119 (APC-Cy7, clone TER-119), CD8 (APC, clone 53-6.7), F4/80 (FITC, clone BM8), CD19 (PerCPCy5.5, clone 6D5), CD3 (PE, clone 17A2), Ly-6C (PECy7, clone HK1.4), Ly-6G (PECy7, clone 1A8), NK1.1 (PECy7, clone PK136), CD45 (AF700, clone 30-F11, Biolegend) and Fixable Viability Dye eFluor 780 (APC-eFluor 780, eBio-science). In case of dendritic cell purification CD11c (PECy7, clone N418, eBioscience), MHCII (PE, MHC Class II (I-A/I-E) Monoclonal Antibody (M5/114.15.2), eBioscience) was used. Cells were stained for 30 min at 4 °C in the dark. 1ml PBS supplemented with 2% BSA was added and suspen-sions were centrifuged at 500g for 5 min at 4 °C. Pellets were resuspended in 300µl PBS supplemented with 2% BSA and filtered over 40µm strainer (SPL Life Sciences), filters were rinsed with 1ml PBS supplemented with 2% BSA. Cells were sorted with a BD FACS-Aria III Fusion instrument into PBS supplemented with 20% BSA.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin accessibility mapping by ATAC-seq was performed as previously described (Buenrostro et al., 2013; Corces et al., 2016), with minor adaptations. After centrifugation, the pellet was carefully resuspended in the transposase reaction mix (12.5µl 2xTD buffer, 2µl TDE1 (Illumina), 10.25µl nuclease-free water, and 0.125µL 10% NP-40 (Sigma) for macrophages and dendritic cells or 0.25µl 1% digitonin (Promega) for all other cell types) for 30 min at 37 °C. Following DNA purification with the MinElute kit eluting in 11µl, 1µl of the eluted DNA was used in a quantitative PCR reaction to estimate the optimum number of amplification cycles.
The remaining 10μl of each library were amplified for the number of cycles corresponding to the Cq value from the qPCR (i.e., the cycle number at which fluorescence has increased above background levels, rounded down). Library amplification was followed by SPRI beads (Beckman Coulter) size selection to exclude fragments larger than 1,200bp. DNA concentration was measured with a Qubit fluorometer (Life Technologies). Library amplification was performed using custom Nextera primers (Buenrostro et al., 2013). The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50bp single-end configuration.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
open chromatin (ATAC-seq)
ATAC-seq sample of spleen CD8 T cell cells and treatment: H for H
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| Data processing |
Raw reads were trimmed with trimmomatic (version 0.32)
aligned to the mouse reference genome (mm10) using bowtie2 (version 2.2.4)
Primary alignments with mapping quality greater than 30 were retained.
ATAC-seq peaks were called using MACS (version 2.7.6 (Zhang et al., 2008)) on each individual sample.
Peaks were aggregated into a list of consensus peaks using the function reduce of the package GenomicRanges (version 1.38.0) in R (version 3.6.1).
Consensus peaks that overlapped with blacklisted genomic regions (downloaded from https://github.com/Boyle-Lab/Blacklist/tree/master/lists) were discarded.
Quantitative measurements were obtained by counting reads within consensus peaks using the function summarizeOverlaps from the GenomicAlignments (version 1.22.1) package in R (version 3.6.1).
Assembly: mm10
Supplementary files format and content: csv file of read counts
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| Submission date |
May 24, 2022 |
| Last update date |
Feb 24, 2024 |
| Contact name |
Christoph Bock |
| E-mail(s) |
cbock@cemm.oeaw.ac.at
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| Organization name |
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences
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| Street address |
Lazarettgasse 14
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| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE204734 |
JAK-STAT pathways maintain homeostasis in immune cells (ATAC-Seq) |
| GSE204736 |
JAK-STAT signaling maintains homeostasis in T cells and macrophages |
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| Relations |
| BioSample |
SAMN28642297 |
| SRA |
SRX15442075 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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