Transcriptome-wide high-throughput mapping of protein-RNA occupancy profiles using POP-seq
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA597062 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10749115.fastq.gz | Fastqsanger.gz | |||
| SRR10749116.fastq.gz | Fastqsanger.gz | |||
| SRR10749117.fastq.gz | Fastqsanger.gz | |||
| SRR10749118.fastq.gz | Fastqsanger.gz | |||
| SRR10749119.fastq.gz | Fastqsanger.gz |
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Project description:Interaction between proteins and RNA is critical for post-transcriptional regulatory processes. Existing high throughput methods based on crosslinking of the protein-RNA complexes and polyA pull down are reported to contribute to biases and are not readily amenable for identifying interaction sites on non polyA RNAs. We present Protein Occupancy Profile-Sequencing (POP-seq), a phase separation based method in three versions, one of which does not require crosslinking, thus providing unbiased protein occupancy profiles on whole cell transcriptome without the requirement of polyA pulldown. Our study demonstrates that ~68% of the total POP-seq peaks exhibited an overlap with publicly available protein-RNA interaction profiles of 97 RNA binding proteins (RBPs) in K562 cells. We show that POP-seq variants consistently capture protein-RNA interaction sites across a broad range of genes including on transcripts encoding for transcription factors (TFs), RNA-Binding Proteins (RBPs) and long non-coding RNAs (lncRNAs). POP-seq identified peaks exhibited a significant enrichment (p value < 2.2e-16) for GWAS SNPs, phenotypic, clinically relevant germline as well as somatic variants reported in cancer genomes, suggesting the prevalence of uncharacterized genomic variation in protein occupied sites on RNA. We demonstrate that the abundance of POP-seq peaks increases with an increase in expression of lncRNAs, suggesting that highly expressed lncRNA are likely to act as sponges for RBPs, contributing to the rewiring of protein-RNA interaction network in cancer cells. Overall, our data supports POP-seq as a robust and cost-effective method that could be applied to primary tissues for mapping global protein occupancies.
2020-06-30 | GSE142460 | GEO
Project description:Interaction between proteins and RNA is critical for post-transcriptional regulatory processes. Existing high throughput methods based on crosslinking of the protein-RNA complexes and poly-A pull down are reported to contribute to biases and are not readily amenable for identifying interaction sites on non poly-A RNAs. We present Protein Occupancy Profile-Sequencing (POP-seq), a phase separation based method in three versions, one of which does not require crosslinking, thus providing unbiased protein occupancy profiles on whole cell transcriptome without the requirement of poly-A pulldown. Our study demonstrates that ~ 68% of the total POP-seq peaks exhibited an overlap with publicly available protein-RNA interaction profiles of 97 RNA binding proteins (RBPs) in K562 cells. We show that POP-seq variants consistently capture protein-RNA interaction sites across a broad range of genes including on transcripts encoding for transcription factors (TFs), RNA-Binding Proteins (RBPs) and long non-coding RNAs (lncRNAs). POP-seq identified peaks exhibited a significant enrichment (p value < 2.2e-16) for GWAS SNPs, phenotypic, clinically relevant germline as well as somatic variants reported in cancer genomes, suggesting the prevalence of uncharacterized genomic variation in protein occupied sites on RNA. We demonstrate that the abundance of POP-seq peaks increases with an increase in expression of lncRNAs, suggesting that highly expressed lncRNA are likely to act as sponges for RBPs, contributing to the rewiring of protein-RNA interaction network in cancer cells. Overall, our data supports POP-seq as a robust and cost-effective method that could be applied to primary tissues for mapping global protein occupancies.
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Project description:DAXX genome binding/occupancy profiling by high throughput ChIP sequencing
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Project description:Pelvic organ prolapse (POP) is a common multifactorial disease in a heterogeneous population of women. Due to this heterogeneity, the underlying molecular mechanisms contributing to the pathogenesis of POP are still unclear. We sought to identify dysregulated pathways by comparing gene expression profiles of prolapsed and non- prolapsed anterior vaginal wall tissue within the same patient. Biopsies were collected from 12 premenopausal women undergoing prolapse surgery (cystocele POP-Q stage ≥ 2). A full thickness anterior vaginal wall sample was taken from the POP site during anterior colporrhaphy. An additional sample was taken from the non-prolapsed apex of the anterior vaginal cuff. Micro-array analysis was performed using whole genome GE 4x44K microarrays. Beside a significance analysis of micro-array (SAM), also a visual cluster analysis was performed. 12 women with POP: 12 biopsies anterior vaginal wall (POP site) versus 12 biopies precervical anterior vaginal wall ( non POP site)
2015-07-31 | E-GEOD-53868 | biostudies-arrayexpress
Project description:Purpose: The goal of this study is to compare DAXX genome binding/occupancy by high throughput ChIP sequencing in control, wild type DAXX overexpressd cells (WT DAXX OE), and wild type DAXX SUMO interacting motif mutant overexpressd cells (DSM OE). Methods: The panel of MDA_MB-231 derived cell lines (control, WT DAXX OE, DSM OE) were cultured in complete DMEM. At about 90% confluency, the cells were crosslinked by adding 37 % formaldehyde to the final concentration of 1% for 10 min at room temperature. Crosslinking was stopped by adding glycine to the final concentration of 125 mM. Cells were lifted, washed with cold PBS, and pelleted by centrifugation. The cells were resuspended in a swelling buffer in the presence of the protease inhibitor cocktail (Sigma) and then pelleted and resuspended in the SDS lysis buffer. The lysates were transferred to a Covaris microTUBE and sonicated with an E220 Covaris Ultrasonicator. Chromatin fragmentation (~500 bps) was verified through agarose gel electrophoresis. The fragmented chromatins were diluted and incubated with a control IgG and the DAXX mAb (5G11) along with protein A/G magnetic beads. The beads were washed sequentially with a low salt buffer, high salt buffer, LiCl buffer, and TE buffer (twice). The immunoprecipitated chromatins were eluted at 65 °C for 15 min, and the eluted chromatins were subjected to proteinase K digestion at 65 °C for 3 h. The DNAs were recovered through a Qiagen mini-prep column. The immunoprecipitated DNAs were used for qPCR and library construction and high throughput sequencing using an Illumina Hi-Seq 2500 sequencer. Results: We surveyed genome-wide occupancy of DAXX using ChIP-seq technology. Overexpression of WT DAXX increased DAXX?s chromatin association specifically with lipogenic pathway genes where DSM OE reverse. Conclusions: Our study represents the first detailed analysis of DAXX occupancy in lipoegenic gene transciption.
2021-12-20 | GSE190783 | GEO
Project description:Pelvic organ prolapse (POP) is a common multifactorial disease in a heterogeneous population of women. Due to this heterogeneity, the underlying molecular mechanisms contributing to the pathogenesis of POP are still unclear. We sought to identify dysregulated pathways by comparing gene expression profiles of prolapsed and non- prolapsed anterior vaginal wall tissue within the same patient. Biopsies were collected from 12 premenopausal women undergoing prolapse surgery (cystocele POP-Q stage ≥ 2). A full thickness anterior vaginal wall sample was taken from the POP site during anterior colporrhaphy. An additional sample was taken from the non-prolapsed apex of the anterior vaginal cuff. Micro-array analysis was performed using whole genome GE 4x44K microarrays. Beside a significance analysis of micro-array (SAM), also a visual cluster analysis was performed.
2015-07-31 | GSE53868 | GEO
Project description:BRG1 occupancy profiling by high throughput sequencing from control and PTEN knockdown 22RV-1 cells
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Project description:Chromatin endogenous cleavage (ChEC) uses fusion of a protein of interest to micrococcal nuclease (MNase) to target calcium-dependent cleavage to specific genomic loci in vivo. Here we report the combination of ChEC with high-throughput sequencing (ChEC-seq) to determine genomic occupancy of the generalist transcription factor Nsi1 and the catalytic subunit of the SWI/SNF chromatin remodelling complex Snf2 in the opportunistic yeast Candida albicans. Time-based analysis of ChEC-seq data reveals two classes of sites for each transcriptional regulator, one exhibiting rapid cleavage during the first 5 min with robust consensus motifs and the second showing slow cleavage at largely unique sites with low-scoring motifs. The ChEC-seq procedure described here will allow a high-resolution genomic location definition which will enable a better understanding of transcriptional regulatory circuits that govern fungal fitness and drug resistance in these medically important fungi.
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Project description:There are a variety of in vivo and in vitro methods to determine the genome-wide specificity of a particular trans-acting factor. However there is an inherent limitation to these candidate approaches. Most biological studies focus on the regulation of particular genes, which are bound by numerous unknown trans-acting factors. Therefore, most biological inquiries would be better addressed by a method that maps all trans-acting factors that bind particular regions rather than identifying all regions bound by a particular trans-acting factor. Here, we present a high-throughput binding assay that returns thousands of unbiased measurements of complex formation on nucleic acid. We applied this method to identify transcriptional complexes that form on DNA regions upstream of genes involved in pluripotency in embryonic stem cells (ES cells) before and after differentiation. The raw binding scores, motif analysis and expression data are used to computationally reconstruct remodeling events returning the identity of the transcription factor(s) most likely to comprise the complex. The most significant remodeling event during ES cell differentiation occurred upstream of the REST gene, a transcriptional repressor that blocks neurogenesis. We also demonstrate how this method can be used to discover RNA elements and discuss applications of screening polymorphisms for allelic differences in binding.
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