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Series GSE50177 Query DataSets for GSE50177
Status Public on Sep 28, 2013
Title Promiscuous RNA binding by PRC2
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Genome binding/occupancy profiling by high throughput sequencing
Summary Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long non-coding RNAs (lncRNAs) recruit PRC2 to chromatin, but the general role of RNA in maintaining repressed chromatin is unknown. ChIP-seq, combined with RNA-seq, indicating that PRC2 is also associated with active genes, but most of these are not regulated by PRC2. These results were complemented by in vitro binding assays measuring the binding constant of human PRC2 to various RNAs and find comparable affinity for human lncRNAs targeted by PRC2 and irrelevant transcripts from ciliates and bacteria. PRC2 binding is size-dependent, with lower affinity for shorter RNAs. These findings support a model in which promiscuous binding of PRC2 to RNA transcripts allows it to scan for target genes that have escaped repression, leading to maintenance of the repressed state. Such RNAs may also provide a decoy for PRC2.
Overall design Cell culture: HEK293T/17 cells were cultured in DMEM with 10% FBS for no longer than 15 passages. ON-TARGETplus SMARTpool for human SUZ12 (Thermo Scientific, Dharmacon cat # L-006957-00-0005) was used to knockdown SUZ12 (siSUZ12) and ON-TARGETplus Non-targeting Pool (Dharmacon cat # D-001810-10-05) was used as a negative control (siCtrl). A total of 25 nM siRNA was transfected in 6-well dishes using Lipofectamine™ RNAiMAX Reagent (Life Technologies, Invitrogen) following the manufacturer’s recommendations. RNA-seq: Polyadenylated RNA was purified from 4 ug of RNA. cDNA libraries were prepared and double-stranded cDNA was fragmented using DNase I according to Illumina specifications, prior to adaptor ligation. Sequencing libraries were amplified and sequenced using an Illumina HiSeq 2000 sequencer. ChIP-seq: 30 million cells at 80% to 90% confluent culture were crosslinked in 1% v/v formaldehyde for 10 min, quenched in 150 mM glycine, washed with cold 1xPBS and harvested by scraping. Cells were lysed in Lysis Buffer (1% w/v SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) with 1x Complete® protease inhibitors (Roche). Cells were sonicated for 10 to 15 min using a Bioruptor™ UCD-200 (Diagenode) with 30 sec pulses at maximum power. Lysates were diluted to 10 ml in IP Buffer (0.01% w/v SDS, 1.1% v/v Triton-X, 1.2 mM EDTA, 16.7 mM Tris-HCL pH 8.0, 167 mM NaCl) and 10 to 20 ng of antibodies were added and incubated overnight at 4 ⁰C with rotation. Antibodies were immunoprecipitated with 60 µl protein G Plus/protein A Agarose Suspension (Calbiochem cat # IP05) and washed sequentially with 1 ml Low Salt Wash Buffer (0.1% w/v SDS, 1% v/v Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), 1 ml High Salt Wash Buffer (0.1% w/v SDS, 1% v/v Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), 1 ml LiCl Wash Buffer (0.25 M LiCl, 1% v/v Nonidet P-40, 1% w/v deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0) and 1 ml TE buffer (Qiagen). DNA was eluted with 0.4 ml of 0.1 M NaHCO3 and 1% w/v SDS. Eluent was transferred into a new tube and NaCl was added to a final concentration of 200 mM. Crosslinks were reversed by incubation at 65 ⁰C for 2 hours. Next, proteins and RNA were digested by adding 33 µl of Digestion Reagent (0.6 M Tris-HCl pH 6.5, 152 mM EDTA, 61 ng/ml RNase A (Invitrogen cat # AM2274) and 0.61 mg/ml Proteinase K (NEB cat # P8102)) following by 1 hour incubation at 37 ⁰C. DNA was extracted by phenol:chloroform and ethanol precipitated. DNA was resuspended in Milli-Q pure water and concentration was measured using Qubit™ (Invitrogen). At least 10 ng of recovered DNA was used to synthesize sequencing libraries using the ChIP-seq Sample Preparation kit (Illumina). Between 6 and 10 pmoles were used for sequencing on the HiSeq2000 sequencer.
Contributor(s) Davidovich C, Cech TR
Citation(s) 24077223
Submission date Aug 25, 2013
Last update date May 15, 2019
Contact name Chen Davidovich
Organization name CU Boulder
Street address 596 UCB, JSCBB, 3415 Colorado Ave
City Boulder
State/province Colorado
ZIP/Postal code 80309
Country USA
Platforms (1)
GPL11154 Illumina HiSeq 2000 (Homo sapiens)
Samples (6)
GSM1215133 EZH2_ChIP-seq
GSM1215134 H3K4me2/3_ChIP-seq
GSM1215135 siCtrl_BiolRep1
BioProject PRJNA217298
SRA SRP029245

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Supplementary file Size Download File type/resource
GSE50177_RAW.tar 910.0 Kb (http)(custom) TAR (of BED)
GSE50177_siSUZ12_vs_siCtrl_HEK293T17.xlsx.gz 2.2 Mb (ftp)(http) XLSX
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Processed data are available on Series record
Processed data provided as supplementary file
Raw data are available in SRA

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