MapR: A Method for Identifying Native R‐Loops Genome Wide

Yan, Qingqing; Sarma, Kavitha

doi:10.1002/cpmb.113

Cited by 19 publications

(22 citation statements)

References 33 publications

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“…In MapR, released R-loops are treated with RNase A and the resultant dsDNA that forms as a consequence of degradation of the RNA within the DNA:RNA hybrid is processed for library preparation and sequencing using standard dsDNA library preparation protocols ( Yan and Sarma, 2020 ). However, non-denaturing bisulfite conversion relies on the presence of ssDNA.…”

Section: Resultsmentioning

confidence: 99%

“…MapR was performed as described ( Yan and Sarma, 2020 ; Yan et al, 2019 ) on 5 × 10 6 cells for BisMapR and control MapR samples (n = 2 replicates per method), with the exception that RNase A was omitted from the stop buffer of the BisMapR sample. Following DNA extraction, the BisMapR samples were bisulfite converted using reagents from the EZ DNA Methylation-Gold Kit (Zymo D5005).…”

Section: Methodsmentioning

confidence: 99%

“…We recently described MapR ( Yan and Sarma, 2020 ; Yan et al, 2019 ), a fast and sensitive R-loop detection strategy founded on the principles of CUT and RUN ( Skene et al, 2018 ; Skene and Henikoff, 2017 ) and the specificity of RNase H for the recognition of DNA:RNA hybrids. In MapR, a catalytically inactive RNase H targets micrococcal nuclease to R-loops to cleave and release them for high-throughput sequencing.…”

Section: Introductionmentioning

confidence: 99%

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A nuclease- and bisulfite-based strategy captures strand-specific R-loops genome-wide

Wulfridge

Sarma

2021

eLife

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R-loops are three-stranded nucleic acid structures with essential roles in many nuclear processes. However, their unchecked accumulation is associated with genome instability and is observed in neurodevelopmental diseases and cancers. Genome-wide profiling of R-loops in normal and diseased cells can help identify locations of pathogenic R-loops and advance efforts to attenuate them. We present an antibody-independent R-loop detection strategy, BisMapR, that combines nuclease-based R-loop isolation with non-denaturing bisulfite chemistry to produce genome-wide profiles that retain strand information. BisMapR achieves greater resolution and is faster than existing strand-specific R-loop profiling strategies. In mouse embryonic stem cells, we apply BisMapR to find that gene promoters form R-loops in both directions and uncover a subset of active enhancers that, despite being bidirectionally transcribed, form R-loops exclusively on one strand. BisMapR reveals a previously unnoticed feature of active enhancers and provides a tool to systematically examine their mechanisms in gene expression.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A nuclease- and bisulfite-based strategy captures strand-specific R-loops genome-wide

Wulfridge

Sarma

2021

eLife

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“…Protein overexpression and purification were performed as described previously (Yan & Sarma, 2020a). Protein overexpression: BL21 (DE3) competent E. coli cells (NEB, Ipswich, MA, Catalog #C2527H) were transformed with 10 ng of either pGEX-6p-1-GST-MNase or pGEX-6p-1-GST-ΔRNH-MNase plasmid (Addgene, Watertown, MA, Catalog #136291 and 136292, respectively).…”

Section: Gst-δrh-mnase and Gst-mnase Protein Purificationmentioning

confidence: 99%

Proper control of R-loop homeostasis is required for maintenance of gene expression and neuronal function during aging

Jauregui-Lozano

Easton

Escobedo

et al. 2021

Preprint

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Age-related loss of cellular function and increased cell death are characteristic hallmarks of aging. While defects in gene expression and RNA metabolism have been linked with age-associated human neuropathies, it is not clear how the changes that occur during aging contribute to loss of gene expression homeostasis. R-loops are DNA-RNA hybrids that typically form co-transcriptionally via annealing of the nascent RNA to the template DNA strand, displacing the non-template DNA strand. Dysregulation of R-loop homeostasis has been associated with both transcriptional impairment and genome instability. Importantly, a growing body of evidence links R-loop accumulation with cellular dysfunction, increased cell death and chronic disease onset. Here, we characterized the R-loop landscape in aging Drosophila melanogaster photoreceptor neurons. Our data shows that transcribed genes in Drosophila photoreceptor neurons accumulate R-loops during aging. Further, our data reveals an association between age-related R-loop accumulation and decreased expression of long and highly expressed genes. Lastly, we show that photoreceptor-specific depletion of Top3β, a DNA/RNA topoisomerase associated with R-loop resolution, leads to both downregulation of of long genes with neuronal function and decreased visual response in flies. Together, our studies present novel data showing increased levels of R-loop in aging photoreceptor neurons, highlighting the link between dysregulation of R-loop homeostasis, gene expression and visual function.

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“…We recently described MapR (Yan and Sarma, 2020;Yan et al, 2019), a fast and sensitive R-loop detection strategy founded on the principles of CUT&RUN (Skene et al, 2018;Skene and Henikoff, 2017) and the specificity of RNase H for the recognition of DNA:RNA hybrids. In MapR, a catalytically inactive RNase H targets micrococcal nuclease to R-loops to cleave and release them for high throughput sequencing.…”

Section: Introductionmentioning

confidence: 99%

BisMapR: a strand-specific, nuclease-based method for genome-wide R-loop detection

Wulfridge

Sarma

2021

Preprint

Self Cite

View full text Add to dashboard Cite

R-loops are three stranded nucleic acid structures with essential roles in many nuclear processes. However, their unchecked accumulation as seen in some neurodevelopmental diseases and cancers and is associated with compromised genome stability. Genome-wide profiling of R-loops in normal cells and their comparison in disease states can help identify precise locations of pathogenic R-loops and advance efforts to attenuate deviant R-loops while preserving biologically important ones. Toward this, we have developed an antibody-independent R-loop detection strategy, BisMapR, that combines nuclease-based R-loop isolation with non-denaturing bisulfite chemistry to produce high-resolution, genome-wide R-loop profiles that retain strand information. Furthermore, BisMapR achieves greater resolution and is faster than existing strand-specific R-loop profiling strategies. We applied BisMapR to reveal discrete R-loop behavior at gene promoters and enhancers. We show that gene promoters exhibiting antisense transcription form R-loops in both directions. and uncover a subset of active enhancers that, despite being bidirectionally transcribed, form R-loops exclusively on one strand. Thus, BisMapR reveals a previously unnoticed feature of active enhancers and provides a tool to systematically examine their mechanisms in gene expression.

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MapR: A Method for Identifying Native R‐Loops Genome Wide

Cited by 19 publications

References 33 publications

A nuclease- and bisulfite-based strategy captures strand-specific R-loops genome-wide

A nuclease- and bisulfite-based strategy captures strand-specific R-loops genome-wide

Proper control of R-loop homeostasis is required for maintenance of gene expression and neuronal function during aging

BisMapR: a strand-specific, nuclease-based method for genome-wide R-loop detection

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