Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-todate lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure.

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodelling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed. However, they still present some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared to existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and emphasizes the role of transcription in the establishment of chromatin structure.RADICL-seq reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency. Application of RADICL-seq to mouse embryonic stem cells (mESCs) and mouse oligodendrocyte progenitor cells (mOPCs) reveals distinct genome occupancy patterns for specific classes of transcripts and uncovers cell-type specific RNA-chromatin interactions.Furthermore, our results highlight the role of transcription in the establishment of the threedimensional (3D) structure of chromatin. Results RADICL-seq technologyWe developed RADICL-seq by using R08, a Mus musculus male embryonic stem cell line with a deeply characterized transcriptome 11 , to identify genome-wide RNA-chromatin (or RNA-DNA) interactions in preserved nuclei (Fig. 1a). We crosslinked cells with two different formaldehyde (FA) concentrations (1% and 2%) to test whether captured interactions were dependent on the amount of crosslinking agent. After crosslinking we isolated the nuclei, partially digested the genomic DNA with DNase I and ends-prepared the chromatin. During technical development of RADICL-seq we evaluated different enzymes that specifically act on RNA to generate a 3'-hydroxyl end, compatible with RNA ligation (Supplementary Fig. 1a). Sequencing data of test RADICL-seq libraries showed that RNase H treatment increased the percentage of uniquely mapped RNA-chromatin interactions by decreasing the ribosomal RNA (rRNA) content, when compared to nuclease S1, RNase V1 or absence of treatment. RNase H enzymatic treatment is known to target RNA-DNA hybrids and, therefore, it could potentially digest nascent RNA bound to its transcription locus, including the highly transcribed rRNA. Indeed, we observed a 2.5-fold reduction in the number of RNA-DNA interactions occurring at a distance below 1 kb between RNAse H-treated and untreated samples (Supplementary Fig. 1b).After enzymatic treatment of the RNA, we introduced a bridge adapter to specifically ligate proximal RNA and DNA (Supplementary Fig. 1c). The adapter is a 5'...

Hi-C is a popular technique to map three-dimensional chromosome conformation. In principle, Hi-C's resolution is only limited by the size of restriction fragments. However, insufficient sequencing depth forces researchers to artificially reduce the resolution of Hi-C matrices at a loss of biological interpretability. We present the Hi-C Interaction Frequency Inference (HIFI) algorithms that accurately estimate restriction-fragment resolution Hi-C matrices by exploiting dependencies between neighboring fragments. Cross-validation experiments and comparisons to 5C data and known regulatory interactions demonstrate HIFI's superiority to existing approaches. In addition, HIFI's restriction-fragment resolution reveals a new role for active regulatory regions in structuring topologically associating domains.