DRIP-seq

DRIP-seq (DRIP-sequencing) is a technology for genome-wide profiling of a type of DNA-RNA hybrid called an "R-loop". DRIP-seq utilizes a sequence-independent but structure-specific antibody for DNA-RNA immunoprecipitation (DRIP) to capture R-loops for massively parallel DNA sequencing.

An R-loop is a three-stranded nucleic acid structure, which consists of a DNA-RNA hybrid duplex and a displaced single stranded DNA (ssDNA). R-loops are predominantly formed in cytosine-rich genomic regions during transcription and are known to be involved with gene expression and immunoglobulin class switching. They have been found in a variety of species, ranging from bacteria to mammals. They are preferentially localized at CpG island promoters in human cells and highly transcribed regions in yeast.

Under abnormal conditions, namely elevated production of DNA-RNA hybrids, R-loops can cause genome instability by exposing single-stranded DNA to endogenous damages exerted by the action of enzymes such as AID and APOBEC, or overexposure to chemically reactive species. Therefore, understanding where and in what circumstances R-loops are formed across the genome is crucial for the better understanding of genome instability. R-loop characterization was initially limited to locus specific approaches. However, upon the arrival of massive parallel sequencing technologies and thereafter derivatives like DRIP-seq, the possibility to investigate entire genomes for R-loops has opened up.

DRIP-seq relies on the high specificity and affinity of the S9.6 monoclonal antibody (mAb) towards DNA-RNA hybrids of various lengths. S9.6 mAb was first created and characterized in 1986 and is currently used for the selective immunoprecipitation of R-loops. Since then, it was used in diverse immunoprecipitation methods for R-loop characterization. The concept behind DRIP-seq is similar to ChIP-sequencing; R-loop fragments are the main immunoprecipitated material in DRIP-seq.

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