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Series GSE227892 Query DataSets for GSE227892
Status Public on May 29, 2023
Title A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB [RIP-seq]
Organism Mus musculus
Experiment type Expression profiling by high throughput sequencing
Summary The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.
 
Overall design RIP-seq
 
Contributor(s) Cherney RE, Mills CA, Herring LE, Braceros AK, Calabrese J
Citation(s) 37066147, 37283223
Submission date Mar 21, 2023
Last update date Sep 08, 2023
Contact name Joseph Mauro Calabrese
Organization name University of North Carolina at Chapel Hill
Department Pharmacology
Lab Calabrese
Street address 120 Mason Farm Road
City Chapel Hill
State/province North Carolina
ZIP/Postal code 27599
Country USA
 
Platforms (1)
GPL19057 Illumina NextSeq 500 (Mus musculus)
Samples (7)
GSM7110375 safb/2 dko clone 13, embyonic stemm cell, safb/2 dko bio rep #1
GSM7110376 safb/2 dko clone 4, embyonic stemm cell, safb/2 dko bio rep #1
GSM7110377 parent wt, embryonic stem cell, wt bio rep #1
This SubSeries is part of SuperSeries:
GSE227893 A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB
Relations
BioProject PRJNA947244

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE227892_ezh2_10kpeaks.bed.gz 147.1 Kb (ftp)(http) BED
GSE227892_ezh2_filtered_peaks.bed.gz 464.7 Kb (ftp)(http) BED
GSE227892_prc2_ezh2peaks_fc.txt.gz 7.9 Mb (ftp)(http) TXT
GSE227892_rip_dko_ezh2_negative_scaled.wig.gz 14.6 Mb (ftp)(http) WIG
GSE227892_rip_dko_ezh2_positive_scaled.wig.gz 15.1 Mb (ftp)(http) WIG
GSE227892_rip_ezh2ko_ezh2_negative_scaled.wig.gz 9.2 Mb (ftp)(http) WIG
GSE227892_rip_ezh2ko_ezh2_positive_scaled.wig.gz 9.6 Mb (ftp)(http) WIG
GSE227892_rip_wt_ezh2_negative_scaled.wig.gz 24.0 Mb (ftp)(http) WIG
GSE227892_rip_wt_ezh2_positive_scaled.wig.gz 24.7 Mb (ftp)(http) WIG
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Raw data are available in SRA
Processed data are available on Series record

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