Optimized ChIP-seq procedure facilitates hormone receptor profiling in human tumors
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE114737 | GEO | 2018/12/27
REPOSITORIES: GEO
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Project description:We developed a general method based on RNA Antisense Purification (RAP) to identify the intermolecular RNA-RNA interactions of a target RNA (RAP-RNA). RAP-RNA identifies endogenous RNA-RNA complexes through in vivo crosslinking, RNA capture with antisense oligonucleotides, and high-throughput RNA sequencing. This approach provides a systematic view of other RNAs that interact with a target RNA, and furthermore can distinguish between direct and indirect RNA-RNA interactions through the use of crosslinking reagents with different reactivities with proteins and nucleic acids. We applied this method to numerous small and large noncoding RNAs, including U1 snRNA, Malat1 lncRNA, Xist lncRNA, U3 snoRNA, U17/Snora73a snoRNA and U12 snRNA. We examined the RNA and chromatin interactions of ncRNAs in mouse embryonic stem cells. We developed and applied three related protocols: RAP-RNA[AMT], RAP-RNA[FA], and RAP-RNA[FA-DSG]. In the RAP-RNA[AMT] protocol, we fixed direct RNA-RNA hybrids in mouse embryonic stem (ES) cells with 4'-aminomethyltrioxalen (AMT), a psoralen-derivative crosslinker; AMT generates inter-strand crosslinks between uridine bases in RNA but does not react with proteins. In the RAP-RNA[FA] protocol, we used a different crosslinking strategy to capture both direct and indirect RNA-RNA interactions: we fixed ES cells using formaldehyde (FA), which crosslinks protein-RNA and protein-protein interactions and thus can capture both indirect interactions as well as direct interactions that are caged or flanked by proteins. In the RAP-RNA[FA-DSG] protocol, we fixed with both FA and disuccinimidyl glutarate (DSG), a strong protein-protein crosslinker, to more efficiently capture RNAs linked indirectly through multiple protein intermediates.
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Project description:The accompanying dataset is the result of a systematic study to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae. We immunopurified, via the use of integrated, C-terminal GFP and 9Myc tags, the five actomyosin motors, Myo1, Myo2, Myo3, Myo4, and Myo5; the kinesin-like proteins Kar3, Kip1, Kip2, Kip3, and Smy1; and the dynein, Dyn1, from S. cerevisiae. Cells were either treated with formaldehyde or with the small molecule latrunculin B. We used formaldehyde crosslinking to stabilize associations between motors proteins and interacting RNAs before IP. Yeast cells growing exponentially in rich medium were treated with formaldehyde, lysed and sonicated, then incubated with magnetic beads coupled to monoclonal antibodies against either 9Myc or GFP to isolate the tagged motor proteins along with any associated RNAs. After IP, the formaldehyde crosslinks were reversed and the enriched RNAs and RNAs purified from the corresponding total lysate were amplified and labeled respectively with Cy5 and Cy3, then jointly hybridized to custom-made, S. cerevisiae oligonucleotide microarrays (Hogan et al., PLoS Biology, 2008). Alternatively, the addition of a low concentration of latrunculin B (2 ug/ml) to live cells to partially solubilize the actin cytoskeleton allowed for successful IP of the motor proteins and associated mRNAs without the need for a chemical crosslinker. In both the case of latrunculin B and formaldehyde treatment, we also performed IPs in which the untagged parent yeast strains were processed for IP and microarray analysis in a manner identical to that of the tagged strains (labeled as mock).
2012-01-27 | GSE35343 | GEO
Project description:The accompanying dataset is the result of a systematic study to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae. We immunopurified, via the use of integrated, C-terminal GFP and 9Myc tags, the five actomyosin motors, Myo1, Myo2, Myo3, Myo4, and Myo5; the kinesin-like proteins Kar3, Kip1, Kip2, Kip3, and Smy1; and the dynein, Dyn1, from S. cerevisiae. Cells were either treated with formaldehyde or with the small molecule latrunculin B. We used formaldehyde crosslinking to stabilize associations between motors proteins and interacting RNAs before IP. Yeast cells growing exponentially in rich medium were treated with formaldehyde, lysed and sonicated, then incubated with magnetic beads coupled to monoclonal antibodies against either 9Myc or GFP to isolate the tagged motor proteins along with any associated RNAs. After IP, the formaldehyde crosslinks were reversed and the enriched RNAs and RNAs purified from the corresponding total lysate were amplified and labeled respectively with Cy5 and Cy3, then jointly hybridized to custom-made, S. cerevisiae oligonucleotide microarrays (Hogan et al., PLoS Biology, 2008). Alternatively, the addition of a low concentration of latrunculin B (2 ug/ml) to live cells to partially solubilize the actin cytoskeleton allowed for successful IP of the motor proteins and associated mRNAs without the need for a chemical crosslinker. In both the case of latrunculin B and formaldehyde treatment, we also performed IPs in which the untagged parent yeast strains were processed for IP and microarray analysis in a manner identical to that of the tagged strains (labeled as mock). transcription profiling
2012-01-27 | E-GEOD-35343 | biostudies-arrayexpress
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2024-07-01 | E-MTAB-14203 | biostudies-arrayexpress
Project description:Mouse embryonic fibroblasts (MEFs) with doxycycline (Dox)-inducible reprogramming cassette MKOS-ires-mOrange and a Nanog-GFP reporter were transduced with lentiviral Dox-inducible Ty1-BFP (blue fluoresce protein with Ty1 tag in the N-terminus) or Hic2-Ty1 (Hic2 with Ty1 tag in the C-terminus) expression vector with MOI 5. One day later reprogramming was initiated by the administration of Dox. 48 hours later, the cells for Hic2 ChIP-seq with anti-Ty1 antibody were crosslinked with 2mM of Disuccinimidyl Glutarate (DSG) for 45 min at room temperature (RT) under constant agitation, before being cross-linked with 1% formaldehyde solution for 10 min at room temperature. For KLF4 ChIP-seq, cells were only cross-linked with 1% formaldehyde solution for 10 min at room temperature. ChIP-seq library was generated with the NEBNext Ultra-II DNA Library Prep kit. Each library was uniquely barcoded using NEBNext Multiplex Oligos for Illumina. Samples were sequenced with the NextSeq High 40PE.
2024-04-26 | E-MTAB-13170 | biostudies-arrayexpress
Project description:Formaldehyde is a widely used fixative in biology and medicine. The current chemical model for formaldehyde cross-linking of proteins is the formation of a methylene bridge that incorporates one carbon atom into the link. Here, we present mass spectrometry data that largely refute this model. Instead, our data show that cross-linking of structured proteins mainly involves a reaction that incorporates two carbon atoms into the link. Under MS/MS fragmentation, the link cleaves symmetrically to yield unusual fragments with a modification of one carbon atom. We apply this new understanding of the underlying cross-linking chemistry to the structural approach of cross-linking coupled to mass spectrometry. First, we cross-linked a mixture of purified proteins with formaldehyde. Our new analysis readily identified tens of cross-links from these proteins, which fit well with their atomic structures. We then perform in-situ cross-linking of human cells in culture. We identified 469 intra-protein and 90 inter-protein cross-links, which also fit well with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. We highlight an example in which formaldehyde cross-links localize the binding site of βNAC on the ribosome. We also find several interactions of actin with auxiliary proteins. Our findings not only expand our understanding of formaldehyde reactivity and toxicity, but also clearly demonstrate how to use this potent reagent for structural studies.
2020-06-25 | PXD015435 | Pride
Project description:BackgroundThe enormous amount of sequence data available in the public domain database has been a gold mine for researchers exploring various themes in life sciences, and hence the quality of such data is of serious concern to researchers. Removal of vector contamination is one of the most significant operations to obtain accurate sequence data containing only a cDNA insert from the basecalls output by an automatic DNA sequencer. Popular bioinformatics programs to accomplish vector trimming include LUCY, cross_match and SeqClean.ResultsIn a recent study, where the program SeqClean was used to remove vector contamination from our test set of EST data compiled through various library construction systems, however, a significant number of errors remained after preliminary trimming. These errors were later almost completely corrected by simply using a re-linearized form of the cloning vector to compare against the target ESTs. The modified trimming procedure for SeqClean was also compared with the trimming efficiency of the other two popular programs, LUCY2, and cross_match. Using SeqClean with a re-linearized form of the cloning vector significantly surpassed the other two programs in all tested conditions, while the performance of the other two programs was not influenced by the modified procedure. Vector contamination in dbEST was also investigated in this study: 2203 out of the 48212 ESTs sampled from dbEST (2007-04-18 freeze) were found to match sequences in UNIVEC.ConclusionVector contamination remains a serious concern to the data quality in the public sequence database nowadays. Based on the results presented here, we feel that our modified procedure with SeqClean should be recommended to all researchers for the task of vector removal from EST or genomic sequences.
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