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Related Experiment Video

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Identification of R-loop-forming Sequences in Drosophila melanogaster Embryos and Tissue Culture Cells Using

Célia Alecki1,2, Nicole J Francis1,2,3

  • 1Institut de recherches cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, QC, H2W 1R7, Canada.

Bio-Protocol
|June 14, 2021
PubMed
Summary

This study presents a new DNA-RNA hybrid immunoprecipitation sequencing (DRIP-seq) protocol for identifying R-loops in Drosophila. The optimized sonication method ensures reproducible results and broad applicability across species.

Keywords:
DRIPDrosophila melanogasterR-loopRNA-DNA hybridS9.6 antibodyTissue culture cells

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Area of Science:

  • Molecular Biology
  • Genomics
  • Epigenetics

Background:

  • R-loops, structures of RNA-DNA hybrids, are implicated in genomic instability and gene regulation.
  • Evidence suggests R-loops play roles in protein targeting and histone modifications.
  • Differential R-loop formation occurs during plant development and is linked to mammalian diseases.

Purpose of the Study:

  • To develop and validate a single-strand DRIP-seq protocol for R-loop identification in Drosophila.
  • To establish a robust and widely applicable method for R-loop mapping.

Main Methods:

  • A novel DRIP-seq protocol utilizing sonication for nucleic acid fragmentation.
  • Application in Drosophila melanogaster embryos and cell culture.
  • Integration of established protocols for high-stringency R-loop identification.

Main Results:

  • The protocol generates homogeneous and highly reproducible nucleic acid fragment pools.
  • Sonication offers advantages over restriction enzymes for fragmentation in DRIP-seq.
  • The method is suitable for de novo characterization of R-loop-forming sequences.

Conclusions:

  • This optimized DRIP-seq protocol provides a reliable tool for R-loop discovery in Drosophila.
  • The sonication-based fragmentation enhances protocol reproducibility and adaptability.
  • The findings support the regulatory role of R-loops and offer a foundation for further research.