Table 2.

The advantages and disadvantages of the methods to investigate protein–RNA interactions.

Method	Advantages	Disadvantages	Analysis of the Interactions Specific to the RNAP II Machinery
RIP	Well-studied Easy-to-use	Dependent on antibody specificity Low signal-to-noise ratio Requires substantial amounts of input materials Does not determine the exact location of RBP-binding sites	Difficult to isolate RNAP II fraction keeping physiological protein–RNA interactions
CLIP irCLIP [54] eCLIP [55] tRIP [56]	Identification of direct protein–RNA interaction sites at single nucleotide resolution High signal-to-noise ratio	Dependent on antibody specificity Low crosslinking efficiency Requires substantial amounts of input materials Complicated procedures	tRIP succeeded in the RNAP II-specific detection Requires further enhancement of detection sensitivity for the precise analysis
KIN-CLIP [59]	High crosslinking efficiency	Requires dedicated devices	Not examined. Requires optimization for less input materials
RNA editing TRIBE [60] STAMP [63]	No need to purify protein–RNA complexes No dependence on crosslinking High detection sensitivity (from single-cell level)	Requires artificial expression of an RBP fused with an RNA-editing enzyme The distribution of detected sites is biased, reflecting the preference of the fused RNA-editing enzyme	Not examined Requires isolation or labeling of RNA specific to the RNAP II machinery
Proximity labeling Proximity-CLIP [69] APEX-RIP [70] CAP-seq [73]	No dependence on crosslinking Efficient isolation of RNA in a specific subcellular fraction	Requires artificial expression of a PL enzyme specific to the fraction of interest Does not identify specific binding sites of an RBP of interest	Not examined Requires development of the labeling strategy specific to the RNAP II machinery