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| Status |
Public on Jun 08, 2020 |
| Title |
ChIP-Seq: ARID1A-KO1 H3K27Ac rep2 |
| Sample type |
SRA |
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| Source name |
endometrial epithelial cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: endometrial epithelial cells
genotype: ARID1A KO
biological replicate: 1
technical replicate: 2
chip antibody: Acetyl-Histone H3 (Lys27) (CST, #8173)
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| Treatment protocol |
Two biological pairs of wild-type (ARID1AWT) and knockout (ARID1AKO) cells were used (PMID: 26953344). These pairs of isogenic ARID1A wildtype and knockout cell lines were established from human endometrial using a CRISPR/Cas9 genome editing method.
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| Growth protocol |
The cell lines used in this study were maintained at 37°C and 5% CO2, and included immortalized isogenic human endometrial epithelial ARID1AWT and ARID1AKO cells. ARID1AWT and ARID1AKO cells were cultured in RPMI 1640 medium supplemented with 15% FBS, 1% MEM non-essential amino acids, and 1% penicillin/streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The chromatin immunoprecipitation (ChIP) assay was performed as previously described on the two independent clones of human endometrial epithelial ARID1AWT and ARID1AKO cells (PMID: 26953344) with the following modifications. Cells were grown in 15 cm dishes, and approximately 1.2×107 cells were cross-linked using Diagenode ChIP cross-link Gold and 1% formaldehyde according to the manufacturer’s instruction. Nuclear contents were extracted using the truChIP Chromatin Shearing kit (Covaris) according to the manufacturer's instructions. Chromatin was sheared for 12 mins in shearing buffer by using a Covaris E220 focused ultrasonicator. The fragment size was ensured to be between 200-600 bp. The sonicated lysates were diluted 5-fold with ChIP dilution buffer (0.1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 1× protease inhibitor), and immunoprecipitated with rotation overnight at 4°C with 0.5-3 µg of antibodies. The antibody/chromatin complex was then precipitated for 3 h by Protein A/G DYNAL magnetic beads (40 µl of 1:1 mixture). Antibody-protein complexes bound to beads were washed once with low salt buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Triton-X100, and 2 mM EDTA), once with high salt buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.1% SDS, 1% Triton-X100, and 2 mM EDTA), once with LiCl buffer (250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl pH 8.0), and twice with TE, pH 8.0. For sequential ChIP, immunocomplex from the first ChIP was eluted with 10 mmol/L dithiothreitol in Tris-EDTA buffer at 37°C and subjected to second round of ChIP. DNA and protein complexes were digested in TE buffer containing 1% SDS, 200 mM NaCl and 1U Proteinase K (Thermo Scientific) at 56°C for 2h, and cross-linking was reversed by heating at 65°C for 4h. DNA fragments were purified using a QIAquick PCR Purification Kit in 55 µl of EB elution buffer.
Tru-seq ChIP-seq library preparation and sequencing using a NextSeq500 platform with single-end reads of 75 bases were performed by JHMI Deep Sequencing and Microarray Core
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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 |
Alignment - For each sample, single-end ChIP-seq reads were aligned to hg19 by Bowtie v0.12.7 (PMID: 19261174), and uniquely mapped reads were selected for analysis, using the parameter option ‘-m 1’. Aligned reads were stored in BAM format using SAMtools v0.1.19 (PMID: 19505943). Peak calling - ChIP-seq peaks were called by MACS2 v2.1.0 (PMID: 18798982) using matched sample and input BAM files. For each transcription factor (ARID1A, BRG1, and RNA Pol II), narrow peaks were called by setting the q-value at 0.01. For histone markers (H3K9me3, H3K27me3, H3K9Ac, and H3K27Ac), broad peaks were called using the broad peak detection mode of MACS2 with a q-value cutoff of 0.05. Peak comparison, annotation, and quantification - ChIP-seq peaks were further compared and annotated using HOMER v4.7.2 (PMID: 20513432). ARID1A consensus peaks and ARID1A/BRG1 consensus peaks were identified by the tool ‘mergePeaks’ considering peaks separated by less than 100 bp as common peaks. We annotated each peak using the ‘annotatePeaks.pl’ function of HOMER. A histogram of read intensities in a 6 kb window around the peak center (option ‘-size 3000 -bin 50’) was generated. For each pair of ARID1AWT and ARID1AKO ChIP-seq experiments, we drew their read intensity histograms based on both ARID1A consensus peaks and ARID1A/BRG1 consensus peaks. PCA analysis - ChIP-seq read coverage was quantified in bedGraph format by MACS2 with ‘-B’ option and further converted to bigwig files by deepTools v2.4.3 (PMID: 24799436). PCA analysis was performed using the plotPCA function. The first 3 principle components were extracted to plot the figure in three dimensions. ChIP-seq intensities in gene regions - The gene region information for hg19 was extracted from the RefSeq annotation file, which was downloaded from the UCSC genome browser (PMID: 25428374), and included 28,830 genes. The ChIP-seq read intensity plots at gene regions were generated by deepTools. In detail, first, ChIP-seq read coverage was quantified using ‘bamCoverage’ with options ‘--normalizeTo1x 2451960000 --ignoreDuplicates --extendReads 200’. Read intensities at gene regions from 3 kb before the transcription start site (TSS) until 3 kb after the transcription end site (TES) were extracted from the read coverage using the ‘scale-region’ command of ‘computeMatrix’. Here, we scaled the length of gene regions to 5 kb. Finally, read intensity curves were plotted by ‘plotProfile’. ChIP-seq intensities in active enhancer regions - First, we identified enhancer marker H3K27Ac peaks in ARID1AWT cells using MACS2 with a q-value cutoff 0.05. As candidate enhancer regions, we selected distal H3K27Ac peaks which were at least 3 kb from the nearest downstream TSS. We limited the distance to 200 kb and identified 13,098 candidate enhancer regions. For each paired ARID1AWT and ARID1AKO ChIP-seq data point, read tag intensity was calculated in a 6 kb window around the center of each enhancer region using the function ‘annotatePeaks.pl’ of HOMER, and further transformed into log2 format for plotting. In the same figure, we plotted three reference lines of fold-change 0.5, 1, and 2.
Genome_build: hg19
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| Submission date |
Jan 02, 2019 |
| Last update date |
Jun 08, 2020 |
| Contact name |
Yohan Suryo Rahmanto |
| E-mail(s) |
suryoysr@gmail.com
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| Organization name |
Johns Hopkins School of Medicine
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| Department |
Department of Pathology
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| Street address |
CRB-2, Rm 376, 1550 Orleans St
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21231 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE106660 |
Inactivation of ARID1A-SWI/SNF Complex Alters Chromatin Compactness at Enhancer Regions and Affects Transcription of Key Tumor Signaling Circuitry [ChIP-Seq] |
| GSE106665 |
Inactivation of ARID1A-SWI/SNF Complex Alters Chromatin Compactness at Enhancer Regions and Affects Transcription of Key Tumor Signaling Circuitry |
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| Relations |
| BioSample |
SAMN10686587 |
| SRA |
SRX5195719 |
| 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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