Mapping RNA–chromatin interactions by sequencing with iMARGI

Abstract

RNA-chromatin interactions represent an important aspect of transcriptional regulation of genes and transposable elements. However, analyses of chromatin-associated RNAs (caRNA) are often limited to one caRNA at a time. Here, we describe the iMARGI (in situ Mapping of RNA-Genome jnteractome) technique used to discover caRNAs and reveal their respective genomic interaction loci. iMARGI starts with in situ crosslinking and genome fragmentation, followed by converting each proximal RNA-DNA pair into an RNA-linker-DNA chimeric sequence. These chimeric sequences are subsequently converted into a sequencing library suitable for paired-end sequencing. A standardized bioinformatic software package called iMARGI-Docker is provided to decode the paired-end sequencing data into caRNA-DNA interactions (https://sysbio.ucsd.edu/ imargi_pipeline). Compared to its predecessor MARGI, in iMARGI the number of input cells is 3-5 million, which is reduced by 100-fold, experimental time is reduced, and clear checkpoints have been established. It takes a few hours a day and a total of 8 days to complete the construction of an iMARGI sequencing library and one day to carry out data processing with iMARGI-Docker.

Stress-induced RNA–chromatin interactions promote endothelial dysfunction

“…Recent technological developments have made it possible to assay DNA-DNA and RNA-chromatin interactions in situ in a genome-wide manner [6][7][8][9][10][11] . Among these tools, in situ mapping of RNA-genome interactome (iMARGI) enables all-RNA-versusthe-genome analyses that can simultaneously identify many caRNAs and their respective genomic interaction loci 7,8 . This feature helped to reveal a large number of caRNAs, including those attached to other chromosomes 7,12 .…”

A New View of Genome Organization Through RNA Directed Interactions

“…Another main limitation in the current protocols and available datasets resides in the relatively small size of the sequenced tags corresponding to the DNA and its interacting RNA (Li et al, 2017 ; Sridhar et al, 2017 ; Bell et al, 2018 ; Quinodoz et al, 2018 ; Wu et al, 2019 ; Bonetti et al, 2020 ; Figure 1D ). Indeed, their short size results in poor mapping of the obtained DNA-RNA pairs to the genome and transcriptome.…”

“…This enabled the authors to determine that transcripts containing TEs are indeed differentially engaged in interactions with chromatin, once again hinting at the importance of TEs in chromatin regulation. Other methodologies, namely MARGI (Sridhar et al, 2017 ) and iMARGI (Wu et al, 2019 ), circumvent this limitation through a protocol that preserves the full length of the respective RNA and DNA tags. This enables the generation of libraries containing longer fragments and results in higher mapping of reads.…”

“…Efficient techniques now include direct ligation of proximal DNA fragments (3C) (Kempfer and Pombo, 2020 ), ligation of barcodes to interacting DNA fragments (SPRITE) (Quinodoz et al, 2018 ), or physical isolation of thin nuclear sections and analysis of the DNA contained within (GAM) (Beagrie et al, 2017 ). The 3C-based methodologies have inspired new protocols to resolve RNA-DNA interactions, such as MARGI (Sridhar et al, 2017 ), GRID-seq (Li et al, 2017 ), CHAR-seq (Bell et al, 2018 ), iMARGI (Wu et al, 2019 ), and RADICL-seq (Bonetti et al, 2020 ). Moreover, SPRITE was also adapted to reveal RNA-DNA interactions (Quinodoz et al, 2018 ; Figure 1D ).…”

Genome-Wide Technologies to Study RNA–Chromatin Interactions

“…In the original MARGI report, the library is sequenced with 100 cycles of paired-end sequencing on an Illumina platform. The authors suggest obtaining 300 million or more read pairs [35]. For GRID-seq, a single-end 100-cycle kit was used for sequencing on an Illumina HiSeq 2500 sequencer.…”