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| Status |
Public on May 11, 2021 |
| Title |
ChIPseq_H3K27ac_S2_2hours_A485 |
| Sample type |
SRA |
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| Source name |
Embryonic
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2
timepoint: 2 hours
treatment+dose: A-485, 1µM
ectopic expression: NA
chip antibody: H3K27ac (Abcam, ab4729)
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| Treatment protocol |
Cells were treated with A-485, A-241, RGFP966, or DMSO at indicated concentrations for indicated time-points.
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| Growth protocol |
Cells were grown in vitro under standard culture conditions.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were washed once in ice-cold PBS prior to crosslinking. For crosslinking, 1/10th volume of fresh formaldehyde solution (11% formaldehyde, 0.05mM EGTA, 1mM EDTA, 100mM NaCl, 50mM Hepes-KOH pH 7.5) was added and incubated for 20 minutes at room temperature with rotation. Crosslinking was quenched by addition of 1/20th volume of 2.5M glycine and incubated for 5 minutes at room temperature with rotation. For isolation of nuclei, cell pellets were washed once in ice-cold PBS and then resuspended in ice-cold nuclear extraction buffer (0.5% NP-40, 2mM EDTA, 10mM NaCl, 20mM Tris-HCl pH 8) and incubated for 5 minutes on ice. Following three sequential incubations in nuclear extraction buffer, cell nuclei were pelleted and resuspended in sonication buffer (0.3% SDS, 1% NP-40, 2mM EDTA, 150mM NaCl, 20mM Tris-HCl pH 7.5) at a concentration equivalent to 50e6 cells per mL. Samples were sonicated in 12x24mm Covaris tubes using the Covaris S2 instrument for 18 minutes using the following settings: 20% Duty Cycle, 1000 cycles/burst, and 10 Intensity. Prior to immunoprecipitation, sheared chromatin was diluted 1:1 in ChIP dilution buffer (1% Triton X-100, 2mM EDTA, 150mM NaCl, 20mM Tris-HCl pH 8) and quantified using Qubit dsDNA HS assay kit. For ChIP-Rx, sheared Drosophila chromatin from S2 cells was spiked into immunoprecipitations at 1:40 ratio of Drosophila:human and processed as a single sample until ChIP-Rx normalization following DNA sequencing. Immunoprecipitations were performed overnight (12-16 hours, 4°C, with rotation) using Protein A and Protein G Dyna beads (Invitrogen) and the indicated antibody. Samples were washed once with ChIP dilution buffer, wash buffer 1 (0.1% SDS, 1% Triton X-100, 2mM EDTA, 500mM NaCl, 20mM Tris-HCl pH 8), wash buffer 2 (0.5% deoxycholate, 0.5% NP-40, 2mM EDTA, 250mM LiCl, 20mM Tris-HCl pH 8), and TE buffer (1mM EDTA, 10mM Tris-HCl pH 7.5) prior to incubation in reverse crosslinking buffer (200mM NaCl, 100mM NaHCO3, 1% SDS, 300μg/mL Proteinase-K) for 4 hours at 55°C with shaking. Finally, the supernatant was reverse-crosslinked overnight (12-16 hours) at 65°C prior to ChIP DNA isolation using Zymogen ChIP DNA Clean and Concentrator Kit (Zymo Research, D5205).
Libraries were generated from ChIP DNA using the NEBNext Ultra II DNA Library Prep Kit (NEB, E7645) and sequenced on an Illumina NextSeq 500 with 75 b.p. single-end reads.
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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 |
Sequencing files were demultiplexed using Bcl2fastq (v2.17.1.14) to generate Fastq files on which QC was performed using FASTQC (v0.11.5).
Sequencing reads were then aligned to custom reference genome consisting the human genome (Hg19) and the Drosophila melanogaster genome (Dm3) using Bowtie2 (v2.3.3).
The resulting SAM files were converted to BAM files using Samtools (v1.4.1) using the view command, which were subsequently sorted, indexed, and potential PCR duplicates removed using the rmdup function.
BAM files were converted into BigWig files using the bamCoverage function (Deeptools, v3.0.0) using the following settings (--normalizeUsing CPM --smoothLength 150 --binSize 50 -e 200 scaleFactor 1).
For experiments with external normalization, the reads mapping to either Hg19 or Dm3 genomes were quantified using FeatureCounts (Subread package, v1.5.0) and the percentage of mapped Dm3 reads as a total of total mapped Dm3+Hg19 reads was calculated. A scale factor was then calculated as the ratio of Dm3 reads in the control treatment condition and the treatment sample, which was then applied as the scaleFactor in the bamCoverage function.
Genome_build: HG19
Supplementary_files_format_and_content: bigWig
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| Submission date |
May 03, 2021 |
| Last update date |
May 11, 2021 |
| Contact name |
Simon J Hogg |
| Organization name |
AbbVie
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| Department |
Oncology Discovery Research
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| Street address |
1000 Gateway Boulevard
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| City |
South San Francisco |
| State/province |
California |
| ZIP/Postal code |
94080 |
| Country |
USA |
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| Platform ID |
GPL19132 |
| Series (2) |
| GSE172051 |
Targeting P300/CBP reveals discrete regulation of transcription and chromatin accessibility by histone acetylation |
| GSE173775 |
Targeting P300/CBP reveals discrete regulation of transcription and chromatin accessibility by histone acetylation [ChIPseq] |
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| Relations |
| BioSample |
SAMN18988580 |
| SRA |
SRX10760659 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5278096_27-CHIP-S-2-A_S9_R1_001.fastq.gz_bowtie2_dm3.sam.sorted.bam.rmdup.bam.cpm.bw |
13.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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