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| Status |
Public on Feb 23, 2021 |
| Title |
H3K27ac_SFTregH2 |
| Sample type |
SRA |
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| Source name |
SF Treg
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| Organism |
Homo sapiens |
| Characteristics |
diagnosis: Juvenile Idiopathic Arthritis
tissue: synovial fluid
cell type: regulatory T cells
chip antibody: anti-histone H3 acetyl K27 antibody (ab4729; Abcam)
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| Growth protocol |
Synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll Isopaque density gradient centrifugation, frozen in 10% DMSO and Treg were sorted after thawing.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each sample, cells were crosslinked with 2% formaldehyde and crosslinking was stopped by adding 0.2 M glycine. Nuclei were isolated in 50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, and 1% Triton X-100 and lysed in 20 mM Tris (pH 7.5), 150mMNaCl, 2mMEDTA, 1% NP-40, 0.3% SDS. Lysates were sheared using Covaris microTUBE (duty cycle 20%, intensity 3, 200 cycles per burst, 60-s cycle time, eight cycles) and diluted in 20 mM Tris (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% X-100. Sheared DNA was incubated overnight with anti-histone H3 acetyl K27 antibody (ab4729; Abcam) pre-coupled to protein A/G magnetic beads. Cells were washed and crosslinking was reversed by adding 1% SDS, 100mM NaHCO3, 200mM NaCl, and 300 µg/ml proteinase K.
DNA was purified using ChIP DNA Clean & Concentrator kit (Zymo Research), end-repair, a-tailing, and ligation of sequence adaptors was done using Truseq nano DNA sample preparation kit (Illumina). Samples were PCR amplified, checked for the proper size range and absence of adaptor dimers, and barcoded libraries were sequenced 75 bp single-end on an Illumina NextSeq500 sequencer.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Reads were mapped to the reference genome hg19 with Bowtie 2.1.0 for H3K27ac ChIP-seq using default settings .
Peaks were called using MACS-2.1.0. Enriched regions were identified compared to the input control using MACS2 callpeak--nomodel --exttsize 300 --gsize=hs -p 1e-9.
Differential binding analysis was performed using DiffBind v1.8.5; TMM was used for read normalization. Enhancer gene associations were determined as the nearest TSS to the center of the enhancer and super-enhancer locus. Super-enhancers were identified by employing the ROSE algorithm using a stitching distance of the MACS2 called peaks of 12.5kb, peaks were excluded that were fully contained in the region spanning 1000bp upstream and downstream of an annotated TSS (-t 1000).
Genome_build: hg19
Supplementary_files_format_and_content: Cod file, containing peak coordinates
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| Submission date |
Aug 18, 2020 |
| Last update date |
Feb 24, 2021 |
| Contact name |
Lisanne Lutter |
| E-mail(s) |
L.Lutter-2@umcutrecht.nl
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| Organization name |
University Medical Center Utrecht
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| Street address |
Lundlaan 6
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| City |
Utrecht |
| ZIP/Postal code |
3584 EA |
| Country |
Netherlands |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE156417 |
Epigenetic profiling to identify a human effector regulatory T cell core signature [H3K27ac] |
| GSE156418 |
Epigenetic profiling to identify a human effector regulatory T cell core signature |
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| Relations |
| BioSample |
SAMN15849362 |
| SRA |
SRX8963359 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4730432_SFTregH2_H3JM5BGXX-_S6.merged.bam_peaks.narrowPeak.gz |
1.1 Mb |
(ftp)(http) |
NARROWPEAK |
| GSM4730432_SFTregH2_H3JM5BGXX-_S6.merged.bam_summits.bed.gz |
648.8 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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