|
| Status |
Public on Sep 20, 2018 |
| Title |
Internally Calibrated ChIP-seq (ICeChIP-seq) using a large panel of antibodies against H3K4 methylations to measure genome-wide histone modification density in mammalian cells |
| Organisms |
Homo sapiens; Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
|
| Summary |
Post-translational modifications (PTMs) on histone proteins regulate genome accessibility and are frequently studied using chromatin immunoprecipitation (ChIP). In ChIP, an antibody putatively specific towards a histone PTM is used to map its genomic locations. ChIP experiments assume perfect antibody-epitope specificity, an assumption previously shown to be problematic, largely through peptide array studies. Among the most well-studied histone PTMs are the mono-, di-, and tri-methylation states of histone H3 lysine 4 (H3K4). While each state has been ascribed different biological functions, the methylation state specificity of antibodies used in these studies has not been systematically interrogated. Here, we use internally calibrated ChIP (ICeChIP) to comprehensively define the specificities of 52 commercially available antibodies marketed to distinguish the three methylation states of H3K4, allowing identification of both high- and low-specificity antibodies. We then conduct ICeChIP-seq with 18 such antibodies of varying specificity. We further find that the sum of H3K4me1 and H3K4me2 across enhancers contacting a promoter correlates strongly with gene expression for all genes, including housekeeping genes, and note that use of low-quality antibodies yields materially different biological interpretations. These results illustrate the way by which variable specificity of commercial antibodies contributes to the “reproducibility crisis” in biological research and demonstrates the need to carefully validate antibodies with techniques appropriate for the intended applications.
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| Overall design |
Semi-synthetic nucleosome standards are spiked into ChIP experiments to conduct internally calibrated ChIP-seq (ICeChIP-seq) with 17 anti-H3K4 methylation antibodies of varying specificities in K562 cells and one anti-H3K4me1 antibody in WT and dCD MLL3/4 R1 mESC cells.
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| Contributor(s) |
Shah RN, Grzybowski AT, Cornett EM, Johnstone AL, Dickson BM, Boone BA, Cheek MA, Cowles MW, Maryanski D, Meiners MJ, Tiedemann RL, Vaughan RM, Arora N, Sun Z, Rothbart SB, Keogh M, Ruthenburg AJ |
| Citation(s) |
30244833, 33872311 |
| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R44 HG008907 |
Internally Calibrated Chromatin Immunoprecipitation Using Barcoded Nucleosomes |
EPICYPHER INC |
Michael-Christopher Keogh |
| R44 HG008907 |
Internally Calibrated Chromatin Immunoprecipitation Using Barcoded Nucleosomes |
EPICYPHER INC |
Zu-Wen Sun |
| R00 CA181343 |
Mechanisms Regulating DNA Methylation Maintenance in Chromatin |
VAN ANDEL RESEARCH INSTITUTE |
Scott Barri Rothbart |
| R35 GM124736 |
Molecular mechanisms of chromatin and epigenetic regulation |
VAN ANDEL RESEARCH INSTITUTE |
Scott Barri Rothbart |
| R01 GM115945 |
Quantitatively probing intra-nucleosomal chromatin variation and function |
UNIVERSITY OF CHICAGO |
Alexander Jackson Ruthenburg |
|
| Submission date |
Sep 06, 2017 |
| Last update date |
Jul 25, 2021 |
| Contact name |
Rohan Nishant Shah |
| E-mail(s) |
rohanshah@uchicago.edu
|
| Organization name |
University of Chicago
|
| Street address |
CLSC 856, 920 E. 58th Street
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platforms (3) |
| GPL15520 |
Illumina MiSeq (Homo sapiens) |
| GPL16417 |
Illumina MiSeq (Mus musculus) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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| Samples (25)
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| Relations |
| BioProject |
PRJNA401866 |
| SRA |
SRP116896 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE103543_AR16_H3K4me1-Corrected_Error_95CI.bedgraph.gz |
6.3 Gb |
(ftp)(http) |
BEDGRAPH |
| GSE103543_AR16_H3K4me1-Corrected_Error_95CI.bigwig |
10.5 Gb |
(ftp)(http) |
BIGWIG |
| GSE103543_AR16_H3K4me1-Corrected_HMD.bedgraph.gz |
6.3 Gb |
(ftp)(http) |
BEDGRAPH |
| GSE103543_AR16_H3K4me1-Corrected_HMD_minus95CI.bedgraph.gz |
6.2 Gb |
(ftp)(http) |
BEDGRAPH |
| GSE103543_AR16_H3K4me1-Corrected_HMD_minus95CI.bigwig |
10.7 Gb |
(ftp)(http) |
BIGWIG |
| GSE103543_AR16_H3K4me1-Corrected_HMD_plus95CI.bedgraph.gz |
6.3 Gb |
(ftp)(http) |
BEDGRAPH |
| GSE103543_AR16_H3K4me1-Corrected_HMD_plus95CI.bigwig |
10.5 Gb |
(ftp)(http) |
BIGWIG |
| GSE103543_AR16_H3K4me1-Corrected_High_Confidence_Peaks.bed.gz |
3.9 Mb |
(ftp)(http) |
BED |
| GSE103543_AR16_H3K4me1_Corrected_HMD.bigwig |
10.6 Gb |
(ftp)(http) |
BIGWIG |
| GSE103543_AR16_H3K4me2-Corrected_Error_95CI.bedgraph.gz |
6.2 Gb |
(ftp)(http) |
BEDGRAPH |
| GSE103543_AR16_H3K4me2-Corrected_Error_95CI.bigwig |
10.5 Gb |
(ftp)(http) |
BIGWIG |
| GSE103543_AR16_H3K4me2-Corrected_HMD.bedgraph.gz |
6.4 Gb |
(ftp)(http) |
BEDGRAPH |
| GSE103543_AR16_H3K4me2-Corrected_HMD_minus95CI.bedgraph.gz |
6.3 Gb |
(ftp)(http) |
BEDGRAPH |
| GSE103543_AR16_H3K4me2-Corrected_HMD_minus95CI.bigwig |
10.7 Gb |
(ftp)(http) |
BIGWIG |
| GSE103543_AR16_H3K4me2-Corrected_HMD_plus95CI.bedgraph.gz |
6.3 Gb |
(ftp)(http) |
BEDGRAPH |
| GSE103543_AR16_H3K4me2-Corrected_HMD_plus95CI.bigwig |
10.6 Gb |
(ftp)(http) |
BIGWIG |
| GSE103543_AR16_H3K4me2-Corrected_High_Confidence_Peaks.bed.gz |
2.9 Mb |
(ftp)(http) |
BED |
| GSE103543_AR16_H3K4me2_Corrected_HMD.bigwig |
10.6 Gb |
(ftp)(http) |
BIGWIG |
| GSE103543_AR16_H3K4me3-Corrected_Error_95CI.bedgraph.gz |
6.2 Gb |
(ftp)(http) |
BEDGRAPH |
| GSE103543_AR16_H3K4me3-Corrected_Error_95CI.bigwig |
10.5 Gb |
(ftp)(http) |
BIGWIG |
| GSE103543_AR16_H3K4me3-Corrected_HMD.bedgraph.gz |
6.5 Gb |
(ftp)(http) |
BEDGRAPH |
| GSE103543_AR16_H3K4me3-Corrected_HMD_minus95CI.bedgraph.gz |
6.3 Gb |
(ftp)(http) |
BEDGRAPH |
| GSE103543_AR16_H3K4me3-Corrected_HMD_minus95CI.bigwig |
10.6 Gb |
(ftp)(http) |
BIGWIG |
| GSE103543_AR16_H3K4me3-Corrected_HMD_plus95CI.bedgraph.gz |
6.3 Gb |
(ftp)(http) |
BEDGRAPH |
| GSE103543_AR16_H3K4me3-Corrected_HMD_plus95CI.bigwig |
10.6 Gb |
(ftp)(http) |
BIGWIG |
| GSE103543_AR16_H3K4me3-Corrected_High_Confidence_Peaks.bed.gz |
672.4 Kb |
(ftp)(http) |
BED |
| GSE103543_AR16_H3K4me3_Corrected_HMD.bigwig |
10.7 Gb |
(ftp)(http) |
BIGWIG |
| GSE103543_RAW.tar |
757.6 Gb |
(http)(custom) |
TAR (of BED, BEDGRAPH, BIGWIG) |
| GSE103543_README_Corrected_files.txt |
1.7 Kb |
(ftp)(http) |
TXT |
| GSE103543_hg38_bar_Ca_1-17.fa.gz |
907.2 Mb |
(ftp)(http) |
FA |
| GSE103543_mm9_bar_Ca_1-17.fa.gz |
819.8 Mb |
(ftp)(http) |
FA |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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