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ISRH-seq: a sensitive and robust methodology for R-loop profiling

ABSTRACT: ISRH-seq: a sensitive and robust methodology for R-loop profiling

PROVIDER: PRJNA971645 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR24517619_1.fastq.gz	Fastqsanger.gz
	SRR24517619_2.fastq.gz	Fastqsanger.gz
	SRR24517620_1.fastq.gz	Fastqsanger.gz
	SRR24517620_2.fastq.gz	Fastqsanger.gz
	SRR24517621_1.fastq.gz	Fastqsanger.gz

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ISRH-seq: a sensitive and robust methodology for R-loop profiling

Project description:R-loops serve as key modulators in various biological processes in humans and plants.Current high-throughput methodologies for R-loop profiling have their own intrinsic pros and cons. Therefore, any robust, sensitive, and amenable methods, complementary and/or advantageous to existing methodologies, are much needed for advancing R-loop biology. We here present an RNase H based cleavage combined with DNA polymerase-mediated in situ biotin-dATP/dCTP labeling of newly synthesized DNA for sequencing, termed ISRH-seq, an antibody-independent approach for strand-specific mapping of R-loops. After conducting a parallel comparison with DRIP-seq, we found that ISRH-seq can identify more R-loop peaks with the same sequencing depth. Moreover, ISRH- and DRIP-specific R-loops had distinct genomic and epigenetic features, exhibited distinct relationships with expression of overlapping genes, and functional divergence. Importantly, we found that ISRH-seq can be scalable down to 250-100ng DNA input, indicative of a robust and sensitive high-throughput methodology. In addition, it can be applied for R-loop profiling under stress conditions. Thus, our study provides a valuable orthogonal methodology for comprehensive profiling of R-loops in plants. It could be easily adapted to other non-plant systems, especially for these biologically important tissues or cell types with limited cell numbers, thereby advancing understanding of R-loop biology.

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Project description:We present in situ DNase I hypersensitivity sequencing (ISDH-seq), a novel methodology for genome-wide open chromatin characterization in plants, which can be applicable to 100-200K nuclei. Compared to conventional DNase-seq and ATAC-seq applied to the same rice tissues, ISDH-seq detects 72% and 120% more open chromatin sites (OCSs), respectively. ISDH-specific OCSs exhibit unique epigenetic signatures: hypomethylation, co-enrichment with H3K27me3, and strong spatial chromatin interaction associations.ISDH-seq overcomes method-specific biases in OCS detection, expands the plant regulatory genome atlas, and offers cross-species applicability. This technology provides critical insights into noncoding regulatory mechanisms and their biological implications in plant genomes.

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aChIP: efficient, sensitive, robust ChIP-seq for economically important plant organs [RNA-Seq]

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aChIP: efficient, sensitive, robust ChIP-seq for economically important plant organs [ChIP-Seq]

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Project description:Background Vitellogenin is a well established biomarker for estrogenic exposure in fish. However, effects on gonadal differentiation at concentrations of estrogen not sufficient to give rise to a measurable vitellogenin response suggest that more sensitive biomarkers would be useful. Induction of zona pellucida genes may be more sensitive but their specificities are not as clear. The objective of this study was to find additional sensitive and robust candidate biomarkers of estrogenic exposure. Results Hepatic mRNA expression profiles were characterized in juvenile rainbow trout exposed to a measured concentration of 0.87 and 10ng ethinylestradiol/L using a salmonid cDNA microarray. The higher concentration was used to guide the subsequent identification of generally more subtle responses at the low concentration not sufficient to induce vitellogenin. A meta-analysis was performed with data from the present study and three similar microarray studies using different fish species and platforms. Within the generated list of presumably robust responses, several well-known estrogen-regulated genes were identified. Two genes, confirmed by quantitative RT-PCR (qPCR), fulfilled both the criteria of high sensitivity and robustness; the induction of the genes encoding zona pellucida protein 3 and a nucleoside diphosphate kinase (nm23). Conclusions The cross-species, cross-platform meta-analysis correctly identified several robust responses. This adds confidence to our approach used for identifying candidate biomarkers. Specifically, we propose that analyses of nm23 together with zona pellucida genes may increase the possibilities to detect an exposure to low levels of estrogenic compounds in fish.

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