RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases.
The impact of R-loops on the physiology and pathology of chromosomes has been demonstrated extensively by chromatin biology research. The progress in this field has been driven by technological advancement of R-loop mapping methods that largely relied on a single approach, DNA-RNA immunoprecipitation (DRIP). Most of the DRIP protocols use the experimental design that was developed by a few laboratories, without paying attention to the potential caveats that might affect the outcome of RNA-DNA hybrid mapping. To assess the accuracy and utility of this technology, we pursued an analytical approach to estimate inherent biases and errors in the DRIP protocol. By performing DRIP-sequencing, qPCR, and receiver operator characteristic (ROC) analysis, we tested the effect of formaldehyde fixation, cell lysis temperature, mode of genome fragmentation, and removal of free RNA on the efficacy of RNA-DNA hybrid detection and implemented workflows that were able to distinguish complex and weak DRIP signals in a noisy background with high confidence. We also show that some of the workflows perform poorly and generate random answers. Furthermore, we found that the most commonly used genome fragmentation method (restriction enzyme digestion) led to the overrepresentation of lengthy DRIP fragments over coding ORFs, and this bias was enhanced at the first exons. Biased genome sampling severely compromised mapping resolution and prevented the assignment of precise biological function to a significant fraction of R-loops. The revised workflow presented herein is established and optimized using objective ROC analyses and provides reproducible and highly specific RNA-DNA hybrid detection.
MTA-DE Momentum, Genome Architecture and Recombination Research Group, Research Centre for Molecular Medicine, University of Debrecen, 4032 Debrecen, Hungary.|Department of Biochemistry and Molecular Biology, University of Debrecen, 4032 Debrecen, Hungary.
László
L
Halász
MTA-DE Momentum, Genome Architecture and Recombination Research Group, Research Centre for Molecular Medicine, University of Debrecen, 4032 Debrecen, Hungary.|Department of Internal Medicine, University of Debrecen, 4032 Debrecen, Hungary.
Zsolt
Z
Karányi
MTA-DE Momentum, Genome Architecture and Recombination Research Group, Research Centre for Molecular Medicine, University of Debrecen, 4032 Debrecen, Hungary.|Department of Biochemistry and Molecular Biology, University of Debrecen, 4032 Debrecen, Hungary.
Beáta
B
Boros-Oláh
MTA-DE Momentum, Genome Architecture and Recombination Research Group, Research Centre for Molecular Medicine, University of Debrecen, 4032 Debrecen, Hungary.|Department of Biochemistry and Molecular Biology, University of Debrecen, 4032 Debrecen, Hungary.
Tímea
T
Kuik-Rózsa
MTA-DE Momentum, Genome Architecture and Recombination Research Group, Research Centre for Molecular Medicine, University of Debrecen, 4032 Debrecen, Hungary.|Department of Biopharmacy, University of Debrecen, 4032 Debrecen, Hungary.
Éva
É
Sipos
MTA-DE Momentum, Genome Architecture and Recombination Research Group, Research Centre for Molecular Medicine, University of Debrecen, 4032 Debrecen, Hungary.
Éva
É
Nagy
MTA-DE Momentum, Genome Architecture and Recombination Research Group, Research Centre for Molecular Medicine, University of Debrecen, 4032 Debrecen, Hungary.|Department of Biochemistry and Molecular Biology, University of Debrecen, 4032 Debrecen, Hungary.
Ágnes
Á
Mosolygó-L
Department of Immunology, University of Debrecen, 4032 Debrecen, Hungary.
Anett
A
Mázló
Department of Immunology, University of Debrecen, 4032 Debrecen, Hungary.
Éva
É
Rajnavölgyi
Department of Biopharmacy, University of Debrecen, 4032 Debrecen, Hungary.
Gábor
G
Halmos
MTA-DE Momentum, Genome Architecture and Recombination Research Group, Research Centre for Molecular Medicine, University of Debrecen, 4032 Debrecen, Hungary.|Department of Biochemistry and Molecular Biology, University of Debrecen, 4032 Debrecen, Hungary.
Lóránt
L
Székvölgyi
D016477:Artifacts; D020029:Base Pairing; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry* / Q000166:cytology / Q000276:immunology; D002874:Chromosome Mapping / Q000379:methods*; D045424:Complex Mixtures / Q000737:chemistry; D004247:DNA / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000737:chemistry; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D019169:Jurkat Cells; D059625:Liquid-Liquid Extraction / Q000379:methods; D009693:Nucleic Acid Hybridization; D061251:Primary Cell Culture; D012313:RNA / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012372:ROC Curve; D052616:Solid Phase Extraction / Q000379:methods
D045424:Complex Mixtures; D005404:Fixatives; D005557:Formaldehyde; D012313:RNA; D004247:DNA; D004262:DNA Restriction Enzymes