mDRIP-seq: a high-throughput and low-cost method for quantitative R-loop profiling in multiple organisms

This study is achieved by Professor Wei Xu’s research team from Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences. The research team established mDRIP-seq, a high-efficiency and low-cost method for R-loop profiling and quantification. R-loop, which plays important roles in many physiological processes, is a three-stranded nucleic acid structure with a DNA:RNA hybridize and a single-stranded DNA strand. Unscheduled R-loops can be formed as genomic threats to trigger DNA replication stress and genome instability, which may contribute to neurodegeneration, cancer, and inflammatory diseases.

The study showed that mDRIP-seq combines a barcoding strategy to pool multiple samples together for DRIP and library preparation with strand-specific sequencing. To barcode samples, the research team established a tailing and single-strand ligation method that utilizes terminal deoxynucleotidyl transferase (TdT) to tail the 3’ terminus of DNA in DNA:RNA hybrids and a short splint to help the barcoded adapter ligate to the tailed hybrids. Compared to exisiting methods, mDRIP-seq requires almost the same dosage of reagents to generate a library for multiple samples as ssDRIP-seq for one sample, with merits of 7-fold less cost and 6-fold less hand-on time per sample. Furthermore, mDRIP-seq could reduce inter-experimental differences in reagent lots, handling personnel, equipment, and capturing times that may introduce batch effects in omics data.

The study showed that mDRIP-seq could simplify R-loop quantitative comparison by using Input read counts for normalization and quantitative assessment of genomic R-loop levels by introducing a large number of RNA:cDNA hybrids synthesized from mRNAs as spike-ins for multiple samples in parallel. Based on mDRIP-seq, the study observed that 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) treatment induced a drop of R-loop signals in the gene body but elevation at the promoter-proximal region of expressed genes. Furthermore, the study demonstrated that different genomic R-loop levels in human cell line HEK293T, mouse cell line NIH/3T3, Arabidopsis wild-type Col-0, rice cultivar 9311, budding yeast strain BY4741, and Escherichia coli strain K12, which may be partly due to the different gene densities among these samples.

To investigate candidate R-loop regulators and their impacts on R-loop formation, the research team applied mDRIP-seq on a large number of budding yeast knockout mutants. The genes in the mutants are related to nucleic acid metabolic processes and chromatin remodeling with enrichment of functions in helicase activity and histone modification activity, which play essential roles in transcriptional regulation and are associated with many diseases in human.Totally, the research team generated an R-loop atlas consisting of more than 200 libraries for nearly 100 gene-deletion mutants, and highlighted patterns of R-loop changes due to gene deletion in yeast at the genome-wide level. Furthermore, the study observed that the changes in protein-coding genes between expression and R-loop signals did not show dramatic linear correlation in mutants such as snf2Δ, snf5Δ, snf6Δ, def1Δ, nam7Δ, dhh1Δ, med1Δ and rad521Δ, indicating that these genes may have R-loop regulatory roles in the expression-independent manner and the underlying mechanisms should be interesting for further investigatation. Therefore, the study supplies the current largest dataset for R-loop profiling and provides valuable resources for further study on roles of these genes in maintenance of R-loop homeostasis.

The team considered that mDRIP-seq can be widely applied to facilitate the research and deepen our understanding of the R-loop biology.

See the article:

mDRIP-seq is a high-throughput method for quantitative profiling of R-loop landscape