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Updated: Aug 25, 2025

Single Nucleotide Polymorphism-sensitive FISH Detection of Locus-specific Ribosomal RNA Transcription in Drosophila melanogaster
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Tissue-Specific, Genome-wide Mapping of R-loops in Drosophila Using MapR.

Juan Jauregui-Lozano1, Kendall Cottingham1, Hana Hall1,2

  • 1Department of Biochemistry, Purdue University, West Lafayette, IN, USA.

Bio-Protocol
|October 17, 2022
PubMed
Summary
This summary is machine-generated.

Researchers developed an improved MapR method to map RNA:DNA hybrids (R-loops) genome-wide in Drosophila. This technique efficiently enriches R-loop DNA from low-input samples, offering a cost-effective solution for studying genomic instability.

Keywords:
BioinformaticsDrosophilaMapRNeuronsR-loop

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Area of Science:

  • Molecular Biology
  • Genomics
  • Epigenetics

Background:

  • R-loops (RNA:DNA hybrids) form during transcription and can cause genomic instability if they accumulate.
  • Existing R-loop mapping methods like DRIP-seq and MapR have limitations, including high input requirements or expensive reagents.
  • Accurate genome-wide R-loop distribution mapping is crucial for understanding cellular homeostasis.

Purpose of the Study:

  • To adapt and optimize the MapR technique for efficient R-loop mapping in low-input Drosophila samples.
  • To demonstrate the utility of an improved MapR protocol incorporating CUT&RUN steps for R-loop enrichment.
  • To provide a cost-effective and accessible method for studying R-loop dynamics.

Main Methods:

  • Purification of GST-tagged, catalytically inactive RNase H1 tethered MapR enzymes (GST-ΔRH-MNase and GST-MNase).
  • Tissue-specific nuclei immuno-enrichment from Drosophila using GFP antibody and magnetic beads.
  • R-loop enrichment via MNase digestion in isolated nuclei followed by DNA purification and library generation.

Main Results:

  • Incorporation of CUT&RUN steps into the MapR protocol significantly improved R-loop-enriched DNA yield from low-input Drosophila nuclei.
  • The optimized MapR method successfully mapped tissue-specific, genome-wide R-loops.
  • The protocol is compatible with low input samples and does not require expensive reagents.

Conclusions:

  • The improved MapR protocol offers a sensitive, cost-effective, and accessible method for genome-wide R-loop mapping.
  • This technique facilitates the study of R-loop roles in various physiological conditions and their contribution to genomic instability.
  • The method is particularly valuable for low-input samples, expanding the scope of R-loop research.