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. Author manuscript; available in PMC: 2020 Mar 1.
Published in final edited form as: Biomed Pharmacother. 2018 Dec 27;111:395–401. doi: 10.1016/j.biopha.2018.12.078

Genome-wide methods for investigating long noncoding RNAs

Mei Cao 1, Jian Zhao 2,*, Guoku Hu 3,*
PMCID: PMC6401243  NIHMSID: NIHMS1518552  PMID: 30594777

Abstract

Long noncoding RNAs (lncRNAs) are large RNA transcripts that do not code for proteins but exert their effects in the form of RNA. To date many thousands of lncRNAs have been identified, their molecular functions and mechanisms of action however are largely unknown. The development of high-throughput experimental technologies, such as ChIRP (Chromatin isolation by RNA purification), CHART (Capture Hybridization Analysis of RNA Targets), RAP (RNA antisense purification), RIP (RNA Immunoprecipitation), CLIP (cross-linking and immunoprecipitation) and RNA pull-down, has led to a rapid expansion of lncRNA research and resulted in many publicly-available databases. This review provides an overview of the current methodologies available for discovering and investigating functions of lncRNAs in various human diseases. A comparison and application of these methods are also included. Finally, this paper surveys current databases containing annotations, interactome networks and functions of lncRNAs. The appropriate use of these methods and databases will provide not only high-resolution functional features of lncRNAs, but also enhance our understanding of the underlying mechanisms by which lncRNAs regulate a variety of biological processes.

Keywords: lncRNA, ChIRP, CHART, RIP, RNA pull-down

1. Introduction

Long noncoding RNAs (lncRNAs) are non-protein coding RNA transcripts that are greater than 200 nucleotides in length [1]. lncRNAs are transcribed from various genomic regions, such as in the enhancers, promoters, introns, anti-sense coding, or intergenic regions of genes [2]. lncRNAs exhibit more highly spatial and temporal restricted expression patterns than mRNAs. Although lncRNAs are present in both the nucleus and the cytoplasm, the fact that many lncRNAs are enriched in the nucleus highlights a broader role for lncRNAs as epigenetic regulators within the nucleus [3]. Indeed, chromatin signature of actively transcribed genes has uncovered around 3,500 lncRNAs in mammals [4, 5]. More recently, over 100 thousands of lncRNAs have been defined in human genome [1, 6]. Unlike protein-coding mRNA molecules, lncRNAs are initially transcribed as primary transcripts by both RNA Polymerase II and III in the nucleus and also can be regulated by transcriptional factors [4, 5]. Multiple lines of evidence demonstrate that lncRNAs exert their functions at the level of chromatin and epigenetics via interaction with RNA binding proteins (RBPs) at specific DNA regions, such as promoters or enhancers [710]. Thus, to better understand the global role of lncRNAs in regulating gene expression, many genomic-context based methodologies have been developed and used for identification of lncRNA binding proteins and chromatin occupancy sites. Indeed, based on their interest, scientists can identify chromatin occupancy sites of a given lncRNA, unknown lncRNAs to which an RBP binds and interacting lncRNAs for a given lncRNA using the omic approaches followed by Next-Generation Sequencing (NGS) of RNA or DNA, and mass spectrometry (MS) (Figure 1).

Figure 1. Schematic representation of high-throughput strategies for lncRNA investigation.

Figure 1.

Crosslinking enables stringent purification of target complexes. AMT specifically crosslinks direct RNA-RNA interactions; FA crosslinks RNA-protein and protein-protein interactions; FA-DSG provides stronger protein-protein crosslinking than FA alone; while UV crosslinks are more specific and only link proteins to RNAs that are in very close proximity. Also see table 1 for a detailed comparison of these methods. ChIRP, chromatin isolation by RNA purification; CHART, Capture Hybridization Analysis of RNA Targets; RAP, RNA antisense purification; RIP, RNA immunoprecipitation; CLIP, cross-linking and immunoprecipitation. FA, formaldehyde; GA, Glutaraldehyde; AMT, 4′-aminomethyltrioxalen; DSG, disuccinimidyl glutarate; UV, ultraviolet.

2. Experimental approaches for lncRNA study in the genomic context

2.1. Hybridization-based approaches (lncRNA–chromatin interactions)

To investigate lncRNA binding sites on chromatin genome-wide, two groups developed similar hybridization-based strategies independently, named ChIRP (Chromatin isolation by RNA purification) [11] and CHART (capture hybridization analysis of RNA targets) [12], and mapped the genomic binding sites for endogenous lncRNAs using those methods in 2011.

ChIRP was developed by Chu et. al., and used to map RNA occupancy at chromatin correlated with a given lncRNA using deep sequencing (ChIRP-seq) [11, 13]. In this method, a pool of 20-nt affinity-probes targeting a specific lncRNA was used to retrieve lncRNA and its binding DNA fragments followed by deep DNA sequencing, thus determining with high-resolution the genomic binding sites for a given lncRNA. To test the reliability of ChIRP, the authors performed ChIRP-seq on endogenous roX2, a lncRNA known to bind to multiple binding sites on the X chromosome in male S2 cells. The results showed that all of the identified 308 roX2 binding sites are on the X chromosome, suggesting that ChIRP-seq is highly sensitive and specific technique for mapping lncRNA occupancy genome-wide at high resolution [11]. More recently, using ChIRP, Olivier-Van Stichelen and Hanover demonstrated that the OGT (O-GlcNAc Transferase) and G6PD (Glucose-6-phosphate dehydrogenase) loci are associated with Xist RNA [14]. Additionally, the enriched RNA, and protein from ChIRP can also be isolated and subjected to RNA and protein analysis [15].

CHART, developed by Simon et. al. [12], was used to purify roX2 lncRNA, its associated proteins and DNA targets in chromatin for the purpose of mapping roX2 RNA binding sites on chromatin. A set of 25-mer desthiobiotin-conjugated-DNA oligonucleotide probes was designed and used for the enrichment of roX2 together with its DNA targets followed by DNA sequencing (CHART-seq). Consistent with the role of roX2 in dosage compensation, the results demonstrated the efficient and specific enrichment of roX2 CHART signals on chromosome X. This technique was further utilized to investigate genome-wide binding locations of Xist lncRNA [8]. CHART-seq, using oligonucleotide probes for Xist, was performed in female mouse cells at four developmental stages: before XCI (X-chromosome inactivation); early-XCI; mid-XCI; and post-XCI. The results demonstrated that Xist density on the X chromosome is increased in a developmental time-dependent manner, and high-resolution maps of Xist binding on the X chromosome across a developmental time course were generated [8].

RNA antisense purification (RAP), developed by Engreitz et. al. in 2013, is another method to map the localization of a given lncRNA across the genome [16]. In this method, a set of 120-nt oligonucleotide affinity-probes (tiled every 15 nucleotides across the sequence of a target lncRNA) was designed and used to capture the probe-RNA-chromatin complexes followed by high-throughput DNA sequencing. Using RAP, the authors demonstrated that Xist binds broadly across the X chromosome and revealed a new mechanism by which Xist orchestrates mammalian XCI by coating and silencing one X chromosome in females [16].

To avoid Type I errors, reduce background and increase specificity, it is important to evaluate the accessibility of probes to target lncRNA before performing experiments. Studies by Simon and colleagues adapted an RNase-H mapping assay strategy [8, 12]. The ability of antisense oligonucleotide probes to reach (access) their target (lncRNA) sites was determined by an RNase H sensitivity assay and only probes with high RNase-H sensitivity were selected for further CHART experiments. This is to ensure that these probes target lncRNA directly. Another strategy used in CHART was to elute probe-RNA-chromatin complexes with RNase-H (specifically hydrolyze RNA that is hybridized to DNA probes) which would release chromatin (combined with probes through lncRNA) from beads and exclude nonspecific binding complexes [12].

2.2. Immunoprecipitation-based approaches (lncRNA–protein interactions)

lncRNAs often exert their function through interaction with RBPs. To identify RNAs bound to a given RBP, immunoprecipitation-based approaches using RBP as bait have been widely used for functional study of lncRNAs. RNA Immunoprecipitation (RIP) is one of the most commonly employed techniques for protein-RNA interaction studies in the last 30 years [17, 18]. The basic principle of RIP is that protein-RNA complexes are immunoprecipitated by a specific antibody against a target protein, such as an RBP. RNAs in these protein-RNA complexes can be purified and analyzed by PCR, microarray analysis (RIP-Chip), or deep sequencing (RIP-seq).

As an example, Zhao et. al. identified Polycomb repressive complex 2 (PRC2)-interacting RNAs in embryonic stem cells (ESCs) using RIP-seq [19]. Antibodies against to EZH2 (one of the four core subunits of PRC2), were used to immunoprecipitate EZH2-binding RNAs followed by Illumina sequencing. This study identified 216 PRC2-interacting lncRNAs in mouse ESCs, including several well-known PRC2-binding lncRNAs (Tsix, RepA, and Xist RNAs), suggesting that the RIP-seq technique is highly sensitive and specific for the detection of protein binding lncRNAs.

Similarly, using a modified RIP-seq approach, the EZH2-associated transcriptome in human gastric cancer cells has been characterized recently by Qi and colleagues [20]. In this study, EZH2-associated RNAs were immunoprecipitated by EZH2-specific antibody from cell nuclei and reverse transcribed using a random primer flanked with a 7-nt barcode sequence. The second cDNA strand was generated using the adaptor ligation strategy instead of template switching strategy, to avoid any bias resulting from guanine preference, followed by Illumina sequencing [20]. Interestingly, the data demonstrated that most of the EZH2-interacting transcripts in gastric cancer cells are mRNAs, suggesting that mRNAs may play a regulatory role in gene expression in the nucleus, considering that cell nuclei were used for the immunoprecipitation in this study.

In another example, Dharap et. al. applied an RIP-Chip approach with antibodies against Sin3A and coREST (corepressors of the RE-1 silencing transcription factor) which are chromatin-modifying proteins (CMPs) [21]. In this work, cortical nuclear lysates from rats subjected to focal ischemia were immunoprecipitated with anti-Sin3A and anti-coREST antibodies. The precipitated RNAs were then purified and subjected to lncRNA microarray analysis. This study demonstrated that 99 Sin3A-enriched lncRNAs and 78 coREST-enriched lncRNAs were significantly increased in the ischemia group compared with the sham group. Among them, the expression of 26 Sin3A-enriched and 11 coREST-enriched lncRNAs was also up-regulated in the ischemic brains, suggesting that stroke-induced lncRNAs may play an important role in epigenetic modifications in the post-ischemic brain through interaction with CMPs [21].

Some variants of RIP have also been established over the past 10 years to precisely map the binding sites of a target protein. Ultraviolet (UV) cross-linking and immunoprecipitation (CLIP) method was originally developed by Ule et. al. in 2003 to purify protein-RNA complexes from mouse brain [22]. Brain tissue was directly irradiated with UV-B light and protein-RNA complexes were immunoprecipitated with Nova antiserum (Nova is one of the earliest identified mammalian tissue-specific splicing factor) [22]. To map the location of Nova binding sites in target RNAs, UVB- irradiated brain lysates were treated with RNase A, to digest the RNA that was not protected by protein. After immunoprecipitation with Nova antiserum, the protein protected RNA fragments were cloned with the use of linker ligation and subjected to sequencing. The results not only identified the known Nova binding sites, but also determined the role of Nova in regulating brain-specific alternative splicing. Thus, CLIP allows precise mapping of protein binding sites on RNAs [22].

HITS-CLIP (also known as CLIP-seq), is a method that combined CLIP with high-throughput sequencing for genome-wide mapping of protein-RNA binding sites in vivo [23]. Using HITS-CLIP, Licatalosi et. al. investigated Nova-binding sites in RNAs and revealed genome-wide biochemical RNA footprints of Nova in multiple mouse brains. Results from this study identified a large number of Nova - RNA interactions in 3’ untranslated regions, suggesting the role of Nova in regulating alternative polyadenylation in the brain [23]. HITS-CLIP provides a robust platform to explore transcriptome-wide binding sites of RBPs.

PAR-CLIP (photoactivatable ribonucleoside–enhanced cross-linking and immunoprecipitation) was first developed by Hafner et. al. in 2010, to determine the binding sites of RBPs and miRNPs (microRNA-containing ribonucleoprotein complexes) at high resolution and transcriptome-wide [24]. In this design, 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) were used to incorporate into nascent RNA transcripts by living cells. The RBP binding sites can be precisely mapped by scoring for thymidine (T) to cytidine (C) transitions in the sequenced cDNA from 4-SU-treated cells; or guanosine (G) to adenosine (A) transitions in the sequenced cDNA from 6-SU-treated cells [25]. Recently, combining PAR-CLIP and anti-m6A immunoprecipitation (MeRIP) approaches, Liu et. al. demonstrated that N6-methyladenosine (m6A) dependent mRNA and lncRNA structural remodeling affects RNA–protein interactions [26]. Furthermore, PAR-CLIP has also been employed to determine RBP binding sites in lncRNAs [24, 25].

Individual-nucleotide resolution CLIP (iCLIP) was modified from CLIP by König et. al. in 2010 and was employed to investigate the RNA splicing maps of heterogeneous nuclear ribonucleoprotein C (hnRNP C) [27]. The aim of iCLIP was to map RBP binding sites at nucleotide resolution. In this study, hnRNP C-RNA complexes were excised from the nitrocellulose membrane, and treated with proteinase to release the RNAs. This also leaves a covalently bound polypeptide fragments at the RNA cross-link sites and causes cDNA premature truncation immediately before the cross-link nucleotide during the reverse transcription. cDNA molecules were then subjected to circularization, linearization, PCR-amplification and high-throughput sequencing. Results from this study revealed single-nucleotide resolution mapping of hnRNP C cross-link sites, suggesting a role for hnRNP particles in splicing regulation [27]. As with all CLIP methods, iCLIP is employed for identifying RBP binding sites on lnRNAs. This includes the interactions of lncRNAs, MALAT1 and NEAT1 with TDP-43, and MALAT1-U2AF65 interaction [28, 29].

2.3. Affinity-based approaches (RNA pull-down)

Biotinylated RNA-protein pull-down followed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) techniques are used to identify interaction proteome of a target RNA. In this method, full length or partial of the target RNA is in vitro synthesized and labeled with biotinylated uridines. After incubation with cellular lysates (or nuclear extracts), biotinylated-RNA-protein complexes are subsequently pulled down with streptavidin beads. Then, RNA-binding proteins are usually separated by SDS-PAGE, and analyzed by MS [3032]. Owing to the use of artificially elevated levels of RNA probe, the advantage of RNA pull-down techniques is to enrich RBPs associated with low abundant target RNA. This method has been widely used for the detection of lncRNA binding proteins in the past 10 years [3335].

Recently, a novel method, Urb-RNA immunoprecipitation (Urb-RIP), was presented by Cottrell and Djuranovic [36]. This method is based on the high-affinity interaction between the sequence-specific RNA stem loop of the MS2 bacteriophage and the RNA recognition motif 1 (RRM1) domain of the “resurrected” snRNA-binding protein Urb. Briefly, 2HA-Urb expressing cells were transfected with a tagged RNA of interest or an untagged control for 24 hours. Then, after UV-irradiation, the cell lysate was incubated with anti-HA beads to isolate 2HA-Urb- tagged RNA-protein complexes. The target RNA associated RBPs and RNAs are subjected to further analysis. The authors confirmed the interaction between polyA-binding protein (PABP) and lncRNA BC200 using Urb-RIP. Thus, Urb-RIP provides another useful tool for lncRNA study as well [36].

Although most of the methods used for investigating lncRNA are adapted from techniques originally developed for mRNA analysis, many improvements in the methods have been made to enable studies on properties and functions of lncRNA, regardless of their abundance. In addition to the modified methods mentioned above, ChIRP-ms [37] is built based on the original ChIRP-seq. Chu and Chang have provided a detailed protocol for this approach [37]. ChIRP-ms offers a comprehensive methodology for the identification of lncRNA-interacting proteins. There is no doubt that more efforts will be spent on the technologies to increase specificity or sensitivity, and to decrease false positives.

3. Employment of the methods in the discovery of disease-associated lncRNAs

The methods described above are powerful approaches for the identification of disease-associated lncRNAs as well as investigation into their acting mechanisms (Table 2). For example, using ChIRP and RIP, Jiang et al. [38] have demonstrated that CCAT1 lncRNA forms a complex with TP63 and SOX2 at the super-enhancers of EGFR, thereby increasing the expression of EGFR and activating both MEK/ERK1/2 and PI3K/AKT signaling pathways, which in turn ultimately promotes the tumorigenesis of squamous cell cancer (SCC). Importantly, knockdown of CCAT1 in the xenograft assays leads to a marked reduction in both volume and mass of the tumors [38], indicating that CCAT1 could be a rational therapeutic target. Recently, employing CHART, Gast et al. [39] have demonstrated the direct interaction between two lncRNAs, MALAT1 and NEAT1, which regulates immune genes and affects the development of atherosclerosis. Interestingly, MALAT1 deficiency causes massive immune-mediated atherosclerosis in ApoE−/− mice suggesting the underlying mechanisms of MALAT1-dependent immune regulation of the cardiovascular system as well as the potential therapeutic applications of this circuit [39]. Applying the RAP approach, Engreitz et al. [40] have demonstrated that U1 small nuclear RNA directly hybridizes to 5’ splice sites and 5’ splice site motifs throughout introns and that MALAT1 interacts with pre-mRNAs indirectly through protein intermediates, suggesting that lncRNAs may target other RNAs as part of their regulatory function. Moreover, using CLIP, Yap et al. [41] have demonstrated that ANRIL lncRNA interacts with CBX7 of the PRC1 complex at the INK4b/ARF/INK4a locus and control senescence. Subsequent studies have demonstrated ANRIL is upregulated in various human tumors, including ovarian cancer [42] and non-small cell lung cancer (NSCLC) [43] indicating that ANRIL could be a biomarker for disease progression.

Table 2.

Examples for the employment of the methods

Method lncRNA Disease Biological function (Targets) Potential clinical application
ChIRP CCAT1 Squamous cell cancer (SCC) Forms a complex with TP63 and SOX2 at the super-enhancers of EGFR [38]. Therapeutic target for SCC
CYTOR Colorectal cancer (CRC) Mediates complex formation between nucleolin and Sam68 [44]. Prognostic biomarker, therapeutic target for CRC
CHART MALAT1, NEAT1 Atherosclerosis MALAT1-NEAT1 interaction regulates immune genes in turn affects the development of atherosclerosis [39]. Therapeutic targets
RAP MALAT1 Hepatocellular carcinoma (HCC) and lung cancer Localizes to nuclear speckles and interacts with multiple serine/arginine RNA splicing proteins and is involved in mRNA processing, splicing, and the export of mRNA [40, 45]. Biomarker for HCC [46, 47] and lung cancer [48]
NORAD Pancreatic cancer and colorectal cancer (CRC) Modulates RBMX and is essential for assembly of a topoisomerase complex and affects cell-cycle progression [49]; serve as sponge for miRNAs [50]. Therapeutic target for pancreatic [51] cancer and CRC[50]
RIP THOR Gastric cancer Binding to SOX9 3’UTR and enhancing its stability [52]. Therapeutic target for gastric cancer
DBH-AS1 Hepatocellular carcinoma (HCC) Activation of FAK/Src/ERK signaling pathway via downregulating miR-138 [53]. Biomarker for HCC and therapeutic target
FOXD2-AS1 Osteoarthritis (OA) Acting as a sponge of miR-206 to modulate CCND1 [54]. Therapeutic target in the treatment of OA.
CLIP ANRIL Epithelial ovarian cancer (EOC); Prostate Cancer; acute lymphoblastic leukemia (ALL) Interacts with CBX7 of the PRC1 complex at the INK4b/ARF/INK4a locus [41]; Regulates P15INK4B and Bcl-2 and inhibits apoptosis and senescence [42]. Biomarker: Prostate Cancer [41]; ALL [55]; ovarian cancer [42]; NSCLC [43]
Thousands of lncRNAs KSHV- and EBV-driven cancers lncRNA - miRNA Interactions [56]. Therapeutic targets
RNA pulldown PINCR human colorectal cancer cells (CRC) Binding to Matrin 3 AT the enhancer regions of p53 target genes [57]. Biomarker and therapeutic target
GAS8-AS1 Hepatocellular carcinoma (HCC) Recruiting the MLL1/WDR5 complex at the promoter of GAS8 [58] Therapeutic target
MANTIS Glioblastoma Interacts with BRG1 and promotes transcription of SOX18, SMAD6, and COUP- TFII [59]. Biomarker and therapeutic target
LINC00152 Lung adenocarcinoma (LAD) Interacts with EZH2 and inhibits IL24 transcription and regulates cell growth and apoptosis [60]. Therapeutic target

4. Conclusions and perspective

As a large number of experiments using the methods mentioned above have been conducted in the investigation of lncRNA interactome networks, millions of interactions between lncRNA, protein and chromatin have been identified. This includes transcriptome-wide binding sites of RBPs, proteome-wide interactions and genome-wide binding sites of target lncRNAs. Indeed, a variety of public databases on lncRNAs have been constructed (Table 3). DIANA-LncBase v2 contains more than 70,000 miRNA:lncRNA interactions and provides an indispensable tool for lncRNA regulation research [61]. NPInter v3.0 holds 491,416 interactions in 188 tissues (or cell lines) from 68 kinds of experimental technologies and allows researchers to predict the function of lncRNAs based on the interactions curated in the database [62]. CLIP-seq data are also annotated with database, CLIPdb [63]. Also, starBase v2.0 is designed to systematically identify RNA-RNA and protein-RNA interaction networks from 108 CLIP-seq [64]. Additionally, an excellent review on lncRNA databases has been published and could provide further insights on this subject [65].

Table 3.

lncRNA Databases

Database # of human lncRNAs or data size Species Description Ref
NONCODE 172,216 17 species An integrated knowledge database of lncRNAs [67]
NPInter 491,416 interactions 22 species Interactions between ncRNAs and biomolecules (proteins, RNAs and DNAs) [62]
lncRNAdb 14,470 68 species Comprehensive annotations of eukaryotic lncRNAs [68]
NRED 1,287 [as of 2008] Human, Mouse Expression data from various platforms and/or studies [69]
lncRNADisease 2,947 lncRNA-disease entries Human lncRNA-disease association database (support prediction) [70]
LNCipedia 107,039 Human A comprehensive compendium of human lncRNAs [6]
ChIPBase ~10,200 ChIP-seq datasets Human, Mouse T ranscription factor occupancy at lncRNA genes [71]
starBase 2.1 million protein-RNA and 1.5 million RNA-RNA interactions Human, Mouse RNA-RNA, and protein-RNA interaction networks from large-scale CLIP-Seq data [64]
DIANA (LncBase) 32,223 Human, Mouse A database of experimentally supported and in silico predicted miRNA Recognition Elements on lncRNAs [61]

Though the application of these methods for studying disease-associated lncRNAs is promising and has resulted in many publicly-available databases, the biological functions of these lncRNAs are still difficult to predict in comparison with protein-coding genes. By utilizing publicly-available genome-wide datasets and computational methods, Signal et al. [66] have developed a pipeline to characterize and predict the functions of lncRNAs. This pipeline is crucial not only for the functional investigation of lncRNAs in silico, but also for informing further experimental strategies.

Taken together, data generated from genomic-context based methodologies will provide not only high-resolution functional features of lncRNAs, but also enhance our understanding of the underlying mechanisms by which lncRNAs regulate a variety of biological processes.

Table 1.

Comparison of the methods

Method Input (Bait) Output (Prey) Endpoint detection Advantage Disadvantage
ChIRP DNA probes (20nt each) for a target lncRNA lncRNA-binding RNAs, genomic elements and proteins RNA/DNA- sequencing and Mass Spectrometry Requires no prior knowledge of the RNA’s structure or functional domains lncRNA sequence information is required and large enough for probe design
CHART DNA probes (25nt each) for a target lncRNA lncRNA-binding RNAs and genomic elements RNA/DNA- sequencing Requires no prior knowledge of the RNA’s structure or functional domains lncRNA sequence information is required and large enough for probe design
RAP >60nt antisense RNA probes for a target lncRNA lncRNA-binding RNAs, proteins and genomic elements RNA/DNA- sequencing and Mass Spectrometry High resolution, specificity, and sensitivity Requires overlapping probes tiled across the entire length of the target RNA to ensure capture even in the case of extensive protein-RNA interactions, RNA secondary structure, or partial RNA degradation
RIP Antibody against a target protein Protein-binding RNAs RNA- sequencing Requires no specialized equipment or reagents Lack of actually protein binding site identification, nonspecific RNA interaction identification, and high signal-to-noise ratio
CLIP Antibody against a target protein Protein-binding RNAs RNA- sequencing Low background noise and high resolution Low sensitivity and possible mutations caused by UV crosslink
RNA pulldown In vitro synthesized biotin-labeled full length or partial of a target RNA lncRNA-binding proteins and RNAs Mass Spectrometry and RNA- sequencing Higher chance to identify weak/transient bindings Artificially elevated levels of lncRNA may result in false positives; difficult to synthesize large lncRNAs (e.g. > 5kb)

Acknowledgements

This work was supported by the National Institutes of Health (DA042704 and DA046831 to GH) and the National Natural Science Foundation of China (31270175 to JZ).

Footnotes

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Conflict of interest

The authors declare no conflicts of interest.

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