Expression profiling by high throughput sequencing Other Third-party reanalysis
Summary
R-Loops are unique RNA-containing chromatin structures, which participate in various key biological processes and associate with multiple human diseases. Accurately and comprehensively profiling R-Loops in the genome is crucial to study their functions under the physiological and pathological conditions. However, the existing methodologies have produced broad discrepancies in R-Loop profiling and an independent strategy is urgently needed. Here, we constructed an artificial DNA-RNA hybrid recognition sensor protein GST-His6-2XHBD with the hybrid-binding domain of RNase H1, and found GST-His6-2XHBD could specifically interact with DNA-RNA hybrids and behaves similarly to the anti-DNA-RNA hybrid S9.6 antibody in DRIPc-seq. Furthermore, we established a convenient method, R-loop CUT&Tag, by combination of GST-His6-2XHBD with a Tn5-based cleavage under targets and tagmentation approach. R-Loop CUT&Tag generates highly specific signals for native R-Loops, and can sensitively detect the R-Loop signals at the promoter, genebody and enhancer. R-Loop CUT&Tag also provides possibilities to genome-widely map the native R-Loops with limited materials, and may benefit the resolving of the broad discrepancies between multiple R-Loop mapping methods.
Overall design
Determination of R-Loops in the human genome with DRIPc-seq, DRIP-seq, and CUT&Tag; Determination of PRO-seq, TT-seq and chromatin-associated RNA-seq in HEK293T cells; Determination of H3K27ac in HEK293T cells. Third-party reanalysis of samples from GSE97072, GSE102474, GSE120637. Related files are linked below.
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