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| Status |
Public on Dec 06, 2016 |
| Title |
WT_H3K4me3 |
| Sample type |
SRA |
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| Source name |
WT_H3K4me3
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| Organism |
Homo sapiens |
| Characteristics |
cell line: H9
cell type: embryonic stem cell (ESC)
genotype/variation: EZH2 +/+ (WT)
chip antibody: H3K4me3 (Abcam, ab8580)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Native ChIP for profiling histone marks was performed as described (Gilfillan et al., 2012) with minor modifications. Cell pellets (from 400,000 cells) were thawed and suspended in 95μl MNase Buffer (50mM Tris-HCl pH8, 1mM CaCl2, 0.2% Triton X-100) supplemented with 5mM Sodium Butyrate and 1X Complete EDTA-free Protease Inhibitor (Roche). Chromatin was digested with micrococcal nuclease (3U; NEB) for 8min at 37°C to obtain predominantly mononucleosomes and dinucleosomes. Stop buffer (10μl of 110mM Tris-HCl pH8, 5mM EDTA) was added to inactivate the reaction. Chromatin was solubilised by brief sonication using a Diagenode Bioruptor for 1min on high power. Chromatin was diluted in 100μl RIPA-IP buffer (280mM NaCl, 1.8% Triton X-100, 0.2% SDS, 0.2% Sodium Deoxycholate, 5mM EGTA supplemented with 5mM Sodium Butyrate and 1X Complete EDTA-free Protease Inhibitor) and insoluble material was removed by centrifugation at 14,000 rpm at 4°C for 15 minutes. Supernatant was transferred to a new tube, 10% was removed for the input sample, and the remaining chromatin was pre-cleared with 50μl pre-washed Protein A and Protein G Dynabeads (Life Technologies) at 4°C for 1 hour. Dynabeads were removed, the pre-cleared chromatin was diluted to 500μl in RIPA-IP buffer and split into 5 tubes. Chromatin was incubated overnight with histone antibodies at 4°C with rotation. Pre-washed Dynabeads (10μl) were added to each tube and incubated at 4°C for 2-3 hours with rotation. Beads were washed five times with RIPA buffer, once with LiCl buffer (250mM LiCl, 10mM Tris-HCl pH8, 0.5% NP-40, 0.5% Sodium Deoxycholate, 1mM EDTA) and once with 1xTE. Beads were suspended in 100μl 1X TE supplemented with 50μg Proteinase K and incubated at 55°C for 1 hour. ChIP and Input DNA were purified using Genomic DNA Clean and Concentrator columns (Zymo) and eluted in 50μl 1xTE. Crosslinked ChIP for profiling EZH2 occupancy was performed using iDeal ChIP-Seq kit for Transcription Factors (Diagenode).
Indexed ChIP-Seq libraries were generated with the NEBNext Master Kit (NEB) using NEBNext Multiplex Oligos for Illumina indexes (NEB).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
WT_H3K4me3_Rep1
WT_H3K4me3_Rep2
ChIP-Seq_RPM.txt
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| Data processing |
ChIP-Seq reads were trimmed using Trim Galore v0.4.0 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) using default parameters to remove the standard Illumina adapter sequence. They were mapped to the human GRCh38 genome assembly using Bowtie 2 v2.2.5 using default parameters. BAM files were imported to Seqmonk and reads were extended by 200bp at their 5’ end to approximate the true insert size.
Processed data files format and content: Quantitated ChIP-Seq data file is tab delimited and shows log2 RPM data for -/+2.5kb surrounding each TSS: (1) mRNA transcript associated with TSS (Ensembl); (2) Chromosome; (3) Start; (4) End; (5) Strand; (6) Associated gene name (Ensembl) (7) mRNA ID (Ensembl); (8) Description (Ensembl); (9-29) log2 RPM value
Libraries were sequenced on the Illumina HiSeq1000/2500 platforms using the default RTA analysis software.
Genome_build: GRCh38
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| Submission date |
Jan 07, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Felix Krueger |
| E-mail(s) |
fkrueger@altoslabs.com
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| Organization name |
Altos Labs
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| Department |
Bioinformatics
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| Street address |
Granta Park
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| City |
Cambridge |
| ZIP/Postal code |
CB21 6GP |
| Country |
United Kingdom |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE76626 |
Characterization of EZH2-deficient human embryonic stem cells [ChIP-seq and bulk RNA-seq] |
| GSE81674 |
Characterisation of EZH2-deficient human embryonic stem cells |
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| Relations |
| BioSample |
SAMN04388017 |
| SRA |
SRX1521178 |
| 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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