Table 2.

Techniques Used to Investigate lncRNAs.

Technique	Bait	Crosslinking	Interaction	Technical Concept	Scope	Reference
nRIP	Protein	No	Direct/indirect	Captures transcriptome and its targets. RNA and protein components associated with the protein of interest.	Genome-wide	75
CLIP-Seq	Protein	UV 254 nm	Direct	Captures protein-RNA interactions in vivo. RNA components associated with protein of interest.	Genome-wide	64
CLIP–mass spectrometry	Protein	UV 254 nm	Direct/indirect	Captures protein-RNA interactions in vivo. Proteins complexes associated with protein of interest and the RNA targets it interacts with.	Genome-wide	126,127
PAR-CLIP	Protein	UV 365 nm	Direct T/C or G/A	Captures protein-RNA covalent binding enabled by efficient crosslinking from 4-SU or 6-SG.	Genome-wide	71,127
iCLIP	Protein	UV 254 nm	Direct; bound to a barcode sequence	Circularization of reverse transcribed products after the ligation of cleavable adaptors.	Genome-wide	68,72
RNA pulldown	lncRNA	Optional	Direct	Special aptamers such as biotin or MS2 fused to the lncRNA pulls down interactome of lncRNA. This includes the targets of lncRNA and complexes interacting. Proteins can be studied by immunoblotting or mass spectrometry.	lncRNA-specific interactions	85,128,129
RAP50,83	Antisense-RNA	Disuccinimidyl glutarate-formaldehyde-aminomethyl-trioxsalen	Direct/indirect	120-nt long nucleotide probes antisense to the target RNA and tiled across the entire RNA target. The probes are biotinylated and captures the lncRNA enrichment amidst protein-RNA interactions, RNA degradations and RNA secondary structures.	Genome-wide	93,130
ChIRP	DNA	Glutaraldehyde	Direct	Antisense DNA probes that hybridize to target RNA. Pulls down endogenous RNA and associated genomic DNA.	Genome-wide	131
ChIRP-domain	DNA	Glutaraldehyde-formaldehyde	Direct	Enables the pulldown of endogenous RNA-chromatin interactions in living cells. Similar to ChIRP, also provides functional information on the architecture and domains of the RNA under investigation.	Genome-wide	88
SHAPE-Seq	RNA	1-Methyl-7 (1M7)–nitroisatoic anhydride (NMIA)	Architecture/structure	The method uses selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), which measures nucleotide resolution flexibility information for RNAs in vitro and in vivo.	Structural information	97
SHAPE-MaP	RNA	1-methyl-7 (1M7)–nitroisatoic anhydride (NMIA)–1M6	Architecture/structure	Similar to SHAPE, but SHAPE-MaP provides additional information on the mutations and yields accurate and high-resolution secondary-structure models and disentangles sequence polymorphisms.	Structural and mutation information	98
DMS-Seq	RNA	Dimethyl sulfate	Architecture/structure	Can be performed in vivo and in vitro. Interacts with unpaired adenine and cytosine residues followed by deep sequencing to identify modifications.	Structural modifications	132
FRAG-Seq	RNA	RNaseP1	Architecture/structure	High-throughput RNA structure probing method that uses high-throughput RNA sequencing of fragments generated by digestion with nuclease P1, which specifically cleaves single-stranded nucleic acids.	Genome-wide	133
PARS, PARTE	RNA	RNase V1, RNase S1	Architecture/structure	High-throughput deep sequencing of RNA fragments that are treated with structure-specific enzymes providing in vitro profiling of secondary structures at single-nucleotide resolution.	Genome-wide	99,134
icSHAPE	RNA	2-methylnicotinic acid imidazolide N3	Architecture/structure	Living cells are treated with the icSHAPE chemical NAI-N3 followed by selective chemical enrichment of NAI-N3–modified RNA, which provides an improved signal-to-noise ratio compared with similar methods leveraging deep sequencing. Purified RNA is then reverse-transcribed to produce cDNA, with SHAPE-modified bases leading to truncated cDNA.	Genome-wide	135,136

4-SU, 4-thiouridine; 6-SG, 6-thiguanosine; cDNA, complementary DNA; ChIRP, chromatin isolation by RNA purification; CLIP, crosslinked immunoprecipitation; CLIP-Seq, crosslinked immunoprecipitation sequencing; iCLIP, individual nucleotide resolution crosslinked immunoprecipitation; DMS-Seq, dimethyl sulfate sequencing; FRAG-Seq, fragmentation sequencing; icSHAPE, in vivo click selective 2′-hydroxyl acylation analyzed by primer extension; lncRNA, long noncoding RNA; nRIP, native RNA immunoprecipitation; PAR-CLIP, photoactivable ribonucleoside-enhanced crosslinked immunoprecipitation; PARS, parallel analysis of RNA structure; PARTE, parallel analysis of RNA structure with temperature elevation; RAP, RNA antisense purification; SHAPE-MaP, selective 2′-hydroxyl acylation analyzed by primer extension mutational profiling; SHAPE-Seq, selective 2′-hydroxyl acylation analyzed by primer extension sequencing; UV, ultraviolet.