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| Status |
Public on Feb 26, 2024 |
| Title |
E14_mESC_reChIP_K4K27_1_R2 |
| Sample type |
SRA |
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| Source name |
E14 mESC
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14 mESC
cell type: embryonic stem cell
strain: E14
genotype: WT
clone: non-clonal
antibody: H3K4me3 (Millipore 07-473) followed by H3K27me3 (CST 9733)
sequencing batch: B
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| Growth protocol |
Cells were cultured at 37 degrees celsius with 5% CO2 on gelatin-coated plates in serum/LIF mESC media (DMEM, 15% FBS, 1x penicillin/streptomycin, 1x NEAA, 1x glutamine, mLIF, beta-mercaptoethanol)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde in prewarmed DMEM for 8 minutes at room temperature. After quenching with 1M glycine, cells were washed by ice cold 1X PBS and resuspend in chromatin resuspension buffer (PBS + 0.5M EDTA). Chromatin pellets were snap-freeze on dry ice and store at -80 degree freezer before use.
ChIP-re-ChIP was performed on 2 million cell pellets. Briefly, cell pellets were gently lysed and treated with MNAse to generate predominantly mononucleosomes then chromatin pre-cleared to reduce non-specific binding by incubating with washed dynabeads for 3 hours at 4 degrees celcius. During this time, three sets of antibody-dynabeads are formed for the IgG control, H3K4me3 and H3K27me3 immunoprecipitations. 5% of the precleared chromatin was set aside as an input control and the remainder split across the three antibody-dynabead reactions for the first overnight immunoprecipitation. Following the first immunoprecipitation, the chromatin-antibody-dynabead complexes are thoroughly washed to remove any non-specific binding, prior to chromatin elution with SDS at 37 degrees. SDS was removed and chromatin concentrated with a buffer exchange using Amicon 3KDa molecular weight filters, setting aside 10% of this sample that represents in-line total H3K4me3 or total H3K27me3 controls. The second immunoprecipitation was then performed overnight using the opposing antibody such that the reChIP was performed in both orientations (H3K4me3 followed by H3K27me3, K4K27 or vice-versa K27K4) with IgG followed by IgG (IgG-IgG) as a negative control. Chromatin is then eluted, treated with RNAse A and proteinase K, crosslinks reversed and enriched DNA fragments purified. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
chromatin immunoprecipitated genomic DNA
Annotated Probe Report for E14_K4K27_peaks.txt
E14-R2-K4K27
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| Data processing |
Single-end reads in fastqs were trimmed (illumina adapters), filtered for quality (phred33 score > 20) and length (>20bp) using TrimGalore v0.6.6 in single-end mode. (trim_galore --phred33 --quality 20 --length 20)
Trimmed reads were then aligned to the mm10 mouse genome (GRCm38.p6) using bwa-mem (bwa v0.7.13) with default parameters. Alignments were then converted to the bam format and idexed using samtools v1.9.
Duplicate alignments were then marked with using MarkDuplicates (picard v2.6.0) and re-indexed with samtools.
bigWig files containing CPM/bp normalised coverage values for each sample were derived from duplicate marked BAM files using bamCoverage (DeepTools v3.5.0) whilst excluding ENCODE blalcklisted genomic regions for the mm10 genome (v2). (bamCoverage --of bigwig -bs 1 -bl mm10-blacklist.v2.bed --normalizeUsing CPM --effectiveGenomeSize 2652783500 --extendReads 147).
Duplicate marked BAM files were loaded into seqmonk v1.48.1 using standard parameters (no deduplication, MAPQ>20, primary alignments only) and extending reads by 200bp.Peak calling for datasets was performed using two biological replicates or clones per ChIP with the IgG-IgG serving as the input control using MACS2 (p-value<1e-5, fragment size 300).
Assembly: GRCm38.p6
Supplementary files format and content: MACS2 peak files (except for IgG-IgG controls)
Supplementary files format and content: CPM/bp normalised bigWig files.
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| Submission date |
Sep 07, 2023 |
| Last update date |
Feb 26, 2024 |
| Contact name |
Janith Seneviratne |
| E-mail(s) |
[email protected]
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| Organization name |
Peter MacCallum Cancer Centre
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| Lab |
Eckersley-Maslin
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| Street address |
Peter MacCallum Cancer Centre, 305 Grattan Street
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| City |
Melbourne |
| State/province |
VIC |
| ZIP/Postal code |
3000 |
| Country |
Australia |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE242686 |
Optimization of mESC ChIP-re-ChIP workflow |
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| Relations |
| BioSample |
SAMN37319504 |
| SRA |
SRX21666878 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7767057_E14-R2-K4K27_dt_bcov_cpm.bw |
309.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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