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Merck

Genomic profiling of native R loops with a DNA-RNA hybrid recognition sensor.

Science advances (2021-02-19)
Kang Wang, Honghong Wang, Conghui Li, Zhinang Yin, Ruijing Xiao, Qiuzi Li, Ying Xiang, Wen Wang, Jian Huang, Liang Chen, Pingping Fang, Kaiwei Liang
RESUMEN

An R loop is a unique triple-stranded structure that participates in multiple key biological processes and is relevant to human diseases. Accurate and comprehensive R loop profiling is a prerequisite for R loops studies. However, current R loop mapping methods generate large discrepancies, therefore an independent method is in urgent need. Here, we establish an independent R loop CUT&Tag (Tn5-based cleavage under targets and tagmentation) method by combining CUT&Tag and GST-His6-2×HBD (glutathione S-transferase-hexahistidine-2× hybrid-binding domain), an artificial DNA-RNA hybrid sensor that specifically recognizes the DNA-RNA hybrids. We demonstrate that the R loop CUT&Tag is sensitive, reproducible, and convenient for native R loop mapping with high resolution, and find that the capture strategies, instead of the specificity of sensors, largely contribute to the disparities among different methods. Together, we provide an independent strategy for genomic profiling of native R loops and help resolve discrepancies among multiple R loop mapping methods.

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Roche
cOmplete, combinación de inhibidores de proteasas sin EDTA, Tablets provided in EASYpacks
Roche
Glucógeno, from mussels
Sigma-Aldrich
Anticuerpo anti-híbrido DN-ARN, clon S9.6, clone S9.6, from mouse
Sigma-Aldrich
4-Thiouridine, ≥98%
Supelco
Poly(ethyleneimine) solution, analytical standard, 50 % (w/v) in H2O
Sigma-Aldrich
Hexarelin, ≥90% (HPLC)
Sera-Mag Magnetic carboxylate modified particles, Cytiva 24152105050350, 1 μm avg. part. size, suspension (5% Solids)