Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1034750 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR26636359_1.fastq.gz | Fastqsanger.gz | |||
| SRR26636359_2.fastq.gz | Fastqsanger.gz | |||
| SRR26636360_1.fastq.gz | Fastqsanger.gz | |||
| SRR26636360_2.fastq.gz | Fastqsanger.gz | |||
| SRR26636361_1.fastq.gz | Fastqsanger.gz |
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Project description:Previously, we introduced an absolute and physical quantitative scale for chromatin immunoprecipitation followed by sequencing. The scale itself was detemined directly from measurements routinely made on sequencing samples without additional reagents or spike-ins. We called this approach sans spike-in quantitative ChIP, or siQ-ChIP. In this paper we extend those results in several ways. First, we simplified the calculations defining the quantitative scale. Second, we highlight the normalization constraint implied by the quantitative scale and introduce a new scheme for generating 'tracks' for siQ-ChIP. We next introduce some whole-genome analyses that are unique to siQ-ChIP which allow us, for example, to project the IP mass onto the genome to evaluate how much of any genomic interval was captured in the IP. We apply these analyses to p300/CBP inhibition and demonstrate that response to inhibition is a function of genomic architecture. In particular, active transcription start sites are only weakly perturbed by p300/CBP inhibition while enhancers are strongly perturbed. Similar observations have been reported in the literature, but without a quantitative scale, those observations have been misinterpreted. We discuss how the siQ-ChIP approach precludes such misinterpretations, which stem from the wide spread community practice of treating unquantified and unnormalized ChIP-seq tracks as though they are quantitative.
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Project description:The RNA-seq data provided here were collected in order to examine the effect of identified coding and non-coding genomic aberrations on gene expression, as part of the publication ‘Whole-genome sequencing of chronic lymphocytic leukemia (CLL) identifies subgroups with distinct biological and clinical features’ by Robbe et al, Nat Gen, 2022.
2022-10-31 | E-MTAB-12124 | biostudies-arrayexpress
Project description:Recently, next-generation sequencing has been introduced as a promising, new platform for assessing the copy number of transcripts, while the existing microarray technology is considered less reliable for absolute, quantitative expression measurements. Nonetheless, so far, results from the two technologies have only been compared based on biological data, leading to the conclusion that, although they are somewhat correlated, expression values differ significantly. Here, we use synthetic RNA samples, resembling human microRNA samples, to find that microarray expression measures actually correlate better with sample RNA content than expression measures obtained from sequencing data. In addition, microarrays appear highly sensitive and perform equivalently to next-generation sequencing in terms of reproducibility and relative ratio quantification.
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Project description:We previously developed sans spike-in quantitative chromatin immunoprecipitation sequencing (siQ-ChIP), a technique that introduces an absolute quantitative scale to ChIP-seq data. The absolute scale is possible because the IP step of ChIP is a competitive binding reaction and produces a classical binding isotherm when antibody or epitope is titrated. The mathematical model that led to siQ-ChIP also predicts that sequencing samples from along an isotherm can reveal differential preferences of the antibody for specific chromatin regions, if the antibody binds with different strengths to different regions. In this paper, we directly test this prediction with several histone post-translational modification (PTM) antibodies. When titrating these antibodies, we identified on- and off-target interactions directly in ChIP-seq. Antibodies that displayed mostly on-target interactions had small, uniform responses for different regions of chromatin, while antibodies that contained off-target interactions had large, variable responses, indicating differences in binding affinity for different chromatin regions. The generation of an isotherm is essential to perform siQ-ChIP analysis. Therefore, we also present a detailed and simplified wet lab protocol, optimized to reproducibly achieve a binding isotherm. We highlight several benchmarks of success that can be used to gain confidence in ChIP experiments, serving as a guide for the practice of siQ-ChIP. Collectively, these studies demonstrate an optimized siQ-ChIP protocol that can be used to determine histone PTM antibody specificity directly in sample chromatin without any exogenous spike-in reagents and at minimal sequencing depth.
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Study Design: N/A , unknown, Other
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Project description:We use Saccharomyces cerevisiae grown on ethanol to perform absolute quantitative multi-omics analysis to map interactions of different cellular processes during the yeast cell cycle.
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Project description:R-loops are dynamic, co-transcriptional nucleic acid structures that facilitate physiological processes and cause DNA damage in certain contexts. Perturbations of transcription or R-loop resolution are expected to change their genomic distribution. Next-generation sequencing approaches to map RNA:DNA hybrids, a component of R-loops, have so far not allowed quantitative comparisons between such conditions. Here we describe quantitative differential RNA:DNA immunoprecipitation (qDRIP), a method combining synthetic RNA:DNA-hybrid internal standards with high-resolution, strand-specific sequencing. We show that qDRIP avoids biases inherent to read-count normalization by accurately profiling signal in regions unaffected by transcription inhibition in human cells, and by facilitating accurate differential peak calling between conditions. Finally, we use these quantitative comparisons to make the first estimates of the absolute count of RNA:DNA hybrids per cell and their half-lives genome-wide. Overall, qDRIP allows for accurate normalization in conditions where R-loops are perturbed and for quantitative measurements that provide previously unattainable biological insights.
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