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| Status |
Public on May 19, 2024 |
| Title |
H3K27ac ChIP-seq in ESC after NVP-2 treatment for 120min treatment rep2 |
| Sample type |
SRA |
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| Source name |
E14 mouse ESC
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14 mouse ESC
cell type: ESC
chip antibody: H3K27ac (Abcam, ab4729)
treatment: NVP-2 100nM for 120min
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| Treatment protocol |
Cells were treated with DMSO or with either 100nM NVP-2 or 1ug/ml Actinomycin D for 120min.
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| Growth protocol |
E14Tg2A Rosa26-Cre Oct4-IRES-Puro mouse ESCs were used for all experiments (Ying et al., 2002). ESCs were cultured in the standard N2B27 medium consisting of 1:1 mix of DMEM/F12 and Neurobasal medium (Thermo Fisher Scientific), supplemented with 1000 units/ml leukemia inhibitory factor (LIF) (Merck Millipore), 1μM PD0325901, 3μM CT-99021 (custom-made by ABCR GmbH, Karlsruhe), 100μM 2-mercaptoethanol, 150μM sodium pyruvate, 0.5x B27 supplement (Thermo Fisher Scientific), 0.5x N2 supplement (made in house or from Thermo Fisher Scientific.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP assay was performed as described previously (Narita et al., 2023) with modifications. mESCs were treated two hours with DMSO, 1 μg/ml Actinomycin D (Tocris Bioscience), or 100 nM NVP2 (Tocris Bioscience), respectively. After treatments, cells were cross-linked with 1% formaldehyde for 10 minutes, neutralized with 0.2 M glycine for 5 minutes, washed twice with PBS, then collected by cell lifter, concentrated as pellets by centrifuged for 10 min at 3000 rpm at 4 °C and stored at -80 °C until use. Cells were resuspended in SDS lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% SDS, 1 mM EDTA, pH 8.0, cOmplete Protease Inhibitor Cocktail (Roche)) and sonicated by a Bioruptor Pico (30 cycles, 30 seconds on and 30 seconds off, Diagenode), then centrifuged for 10 min at 13,000×g at 20 °C for debris removal. The supernatants were stored at -80 °C. The aliquots of the supernatant were 5-fold diluted with ChIP dilution buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, cOmplete Protease Inhibitor Cocktail), incubated 20 hours at 65 °C to reverse cross-linking, then treated with 0.2 mg/ml RNase A (Thermo Fisher Scientific) for 30 min at 37 °C and proteinase K (Thermo Fisher Scientific) for 1 hour at 55 °C. Then DNA fragments were isolated by phenol-chloroform extraction and recovered by ethanol precipitation with glycogen (Thermo Fisher Scientific). DNA fragments were quantified by Nanodrop (Thermo Fisher Scientific). The 300 bp average size of DNA was measured by 4200 TapeStation System (Agilent Technologies). For immunoprecipitation, the supernatants equivalent to 100 μg of mESC chromatin DNA combined with 10 μg of chromatin DNA derived from HEK293T as spike-in were incubated with anti-histone H3 acetyl K27 antibody (ab4729 Abcam) conjugated with Dynabeads M-280 sheep anti-Rabbit IgG (Thermo Fisher Scientific) by rotating overnight 4 °C . The immunoprecipitated Protein-DNA complexes were washed with ChIP dilution buffer, wash buffer high-salt (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), wash buffer low-salt (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% sodium deoxycholate, 0.5% NP-40) once respectively, washed with TE three times, then eluted with elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS). The immunoprecipitated DNAs were reversed cross-link and recovered from the eluates as described above.
ChIP-seq, library was prepared from 20 ng of ChIPed DNA measured by Qubit dsDNA HS Assay (Thermo Fisher Scientific) using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s instructions. Adoptor-ligated, U-excisioned DNA was cleaned by AMPure XP beads (Beckman Coulter) amplified by PCR 6 cycles, then cleaned again and size-selectioned (350-400 bp average size measured by Tapestation). 4 nM pooled Library DNAs were sequenced on a NextSeq 2000 Sequencer (Illumina) as single-end 138-bp reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 2000 |
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| Description |
Human HEK293 cell was used as spike-in.
ESC_Ctrl_TC0_H3K27ac.ab4729_CC39.CC44_peak.bed
ESC_NVP2_100nM_TC120_H3K27ac.ab4729_spike.HEK293_CC39.CC44.spTotal.norm.smooth.bw
ESC_Ctrl_TC0_H3K27ac.ab4729_CC39.CC44_peak.bed
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| Data processing |
Adaptors were trimmed using Cutadapt. Read sequences were aligned to the combined mouse mm10 and human hg19 genome using bwa meme (version 1.0.4) with soft clipping option for supplementary alignments. Duplicated reads were annotated and removed using Picard toolkit tools (version 2.9.1, “Picard Toolkit.” 2019. Broad Institute, GitHub Repository. https://broadinstitute.github.io/picard/; Broad Institute). Low-mapping quality reads (MAPQ < 10) were excluded using samtools (version 1.4). Peak calling was conducted using LanceOtron with the default model (wide-and-deep_jan-2021). The peaks proximal within 2kb are merged using Bedtools93. Peak heights were calculated using bamCompare94 with the following parameters (centerReads, minMappingQuality 10, bin size 20b, smoothing length 400b, extension of reads to 200b, rpm normalization, and input rpm value is subtracted). The peak summit was defined as the center of the 20b bin at maximum height in each peak region. Poorly enriched peaks of maximum peak height at 20bp bin < 8 reads mapped per million (rpm) or read enrichment at +/- 500bp around peak summit center < 1 rpm after input subtraction were excluded. H3K27ac peaks that were not proximal to ATAC-seq peak summit within 400bp, or whose peak summits were more than 1kb from ATAC-seq peak summit were filtered out. The H3K27ac peak changes were quantified by analyzing the read count mapped to peak summit +/- 500bp region. The FC ratios were calculated by using DESeq2 with incorporating scaling factors determined from the HEK293 cell spike-in reads counts.
Assembly: mm10, T2T-CHM13v2.0
Supplementary files format and content: Gene track bigwig files and H3K27ac peak bed file.
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| Submission date |
Mar 05, 2024 |
| Last update date |
May 19, 2024 |
| Contact name |
Chunarum Choudhary |
| E-mail(s) |
chuna.choudhary@cpr.ku.dk
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| Organization name |
University of Copenhagen
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| Department |
Novo Nordisk Foundation Center for Protein Research
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| Lab |
Choudhary Group
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| Street address |
Blegdamsvej 3B
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| City |
Copenhagen N |
| State/province |
--- Select a state --- |
| ZIP/Postal code |
2200 |
| Country |
Denmark |
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| Platform ID |
GPL30172 |
| Series (2) |
| GSE260922 |
Acetylation is not mere a consequence of transcription: histones are acetylated without ongoing transcription [ChIP-seq] |
| GSE260969 |
Acetylation is not mere a consequence of transcription: histones are acetylated without ongoing transcription |
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| Relations |
| BioSample |
SAMN40268934 |
| SRA |
SRX23842166 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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