iMARGI Protocol v1

In situ mapping of RNA-Genome Interactome (iMARGI) is a technique that globally maps native RNA-genome interactions from unperturbed cells. It is an improved version of traditional MARGI published in 2017. Compared to traditional MARGI, this technique performs RNA-DNA proximity ligation in situ inside intact nucleus instead of in solution to reduce random ligation. It also reduces the number of cell input by 100-fold and shortens experimental processing time. In this protocol, cells are crosslinked with formaldehyde. Intact nuclei are collected from fixed cells and permeabilized with SDS. A restriction enzyme AluI is used to digest chromatin into DNA fragments and RNase I is used to fragment RNA. A specially designed biotinylated linker is introduced to ligate to 3´-end of RNA first and then this RNA-ligated linker is ligated to DNA. These ligation steps are controlled by the configuration of the linker and by sequential applications of different end modifications and ligation enzymes. Nuclei are lysed, and successfully ligated products, in the form of RNA-linker-DNA are selected with streptavidin beads, followed by converting the RNA part of chimera into cDNA on beads. cDNA-linker-DNA is circularized and then cut in the middle, producing DNAs with the configuration left.half.Linker-RNA-DNA-right.half.Linker, which are subjected to library amplification and paired-end sequencing.