1a, we first performed in situ RNA ligation and then extended the DNA primer in the linker into ligated RNA with reverse transcriptase. The linker was pre-adenylated at the 5′ end of the RNA and characterized in vitro and in the cell ( Extended Data Fig. We designed a biotin-labeled bivalent linker consisting of a single-stranded RNA (ssRNA) portion for ligation to RNA and a double-stranded DNA (dsDNA) portion for ligation to DNA ( Extended Data Fig. To this end, we stabilized RNAs on chromatin by double fixing cells with disuccinimidyl glutarate (DSG) and formaldehyde, isolated nuclei, and performed in situ DNA digestion with a frequent 4-base cutter AluI. We first chose a triple negative breast cancer MDA-MB-231 cell line to develop an unbiased strategy to map RNA-chromatin interactions genome-wide. Assuming that most interactions represent a physical proximity between the site of transcription and the distal binding site, this comprehensive RNA-chromatin interactome permitted the identification of transcription activity-associated promoter-enhancer interactions both within and beyond TADs. We discovered a large set of both coding mRNAs and ncRNAs that bind to active promoters and enhancers, especially super-enhancers. Application of GRID-seq to two human cell lines (MDA-MB-231 and MM.1S), one mouse cell line (mESC), and one Drosophila cell line (S2), exposed distinct classes of cis- and trans-chromosomal interacting RNAs that were linked to cell type-specific gene expression programs. Here we report a strategy for mapping Global RNA Interactions with DNA by deep sequencing (GRID-seq) that uses a bivalent linker to ligate RNA to DNA in in situ on fixed nuclei. To address these questions, we sought to develop a general approach for comprehensively localizing all potential chromatin-interacting RNAs in an unbiased fashion. However, as these techniques detect both regulatory and static physical interactions that are largely confined within cell type-independent topologically associating domains (TADs) 13, 14, chromatin-associated RNAs may help define chromatin interactions that are directly linked to transcriptional activities and differentiate super-enhancers from typical enhancers 15– 17. The chromatin structure has been analyzed with Hi-C, which detects all possible DNA-DNA interactions 8, 9, and ChIA-PET, which enriches specific factor-mediated interactions 10– 12. RNAs might also play a role in coordinating functional DNA elements in regulated gene expression. However, these methods only allow analysis of one known RNA at a time, and consequently, a global view is lacking on all potential RNA-chromatin interactions, which is critical for addressing a wide range of functional genomics questions. These include Chromatin Isolation by RNA Purification (ChIRP) 5, Capture Hybridization Analysis of RNA Targets (CHART) 6, and RNA Affinity Purification (RAP-DNA) 7, which all rely on using complementary sequences to capture a specific RNA followed by deep sequencing to identify chromatin targets. Various techniques have been developed to localize specific RNAs on chromatin. These findings suggest a role of specific RNA-chromatin interactions in regulating gene expression. Some ncRNAs may mediate genomic interactions predominantly in cis, whereas others, such as MALAT1 and NEAT1, are capable of extensively acting in trans 4. Many ncRNAs appear to act directly on chromatin, as exemplified by various characterized long non-coding RNAs (lncRNAs) 2, 3. Mammalian genomes express not only protein-coding mRNAs but also a large repertoire of non-coding RNAs (ncRNAs) that have regulatory functions in different layers of gene expression. Recent genomic research has revealed that mammalian genomes are more prevalently transcribed than previously thought 1.
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