Table 1.

Genome-wide methods to study translation.

	RNA isolation methodology	Novelty/advantages	Limitations	Reference
Polysome profiling	Purification of polysome-associated mRNAs by centrifugation through a sucrose gradient	Original method to examine translation status of transcriptome	Labor intensive; scaling issues; does not differentiate between active and stalled ribosomes	Zong et al. (1999)
TRAP	Immunoprecipitation (IP) of EFGP-L10a-associated mRNAs from mouse brain tissue	Examines polysome-associated mRNAs within a specific cell type in vivo	Each bacTRAP mouse line is limited to surveying one cell type; EGFP antibodies are costly relative to anti-HA antibody; does not differentiate between active and stalled ribosomes	Heiman et al. (2008), Doyle et al. (2008)
RiboTag	IP of Rlp22-HA-associated mRNAs from mouse tissue	Examines polysome-associated mRNAs within a specific cell type in vivo; takes advantage of Cre recombinase-expressing mouse lines to expand the range of cell types that can be investigated; commercial anti-HA antibody is less costly than in-house EGFP (see TRAP)	Does not differentiate between active and stalled ribosomes	Sanz et al. (2009)
Ribosome profiling	Nuclease digestion of polysome complexes, followed by centrifugation through a sucrose gradient or cushion to purify ribosome-mRNA complexes; ribosome-protected fragments are deep sequenced	Determines ribosome position and translation efficiency for individual mRNAs; reveals novel translational regulatory features (e.g., uORFs, start and termination sites, ribosome stall position)	May be difficult to apply to mouse models	Ingolia et al. (2009), Ingolia et al. (2011)
CLIP	UV-mediated crosslinking of mRNA-protein complexes, followed by nuclease digestion and IP of RBP of interest to recover RBP-protected mRNA fragments	Demonstrated the feasibility of crosslinking mRNA and protein using UV irradiation, which results in covalent bonds	Generated a limited dataset with a high false positive rate; low crosslinking efficiency	Ule et al. (2003)
CLIP-seq or HITS-CLIP	CLIP coupled with deep sequencing	Identifies direct RBP binding sites at nucleotide resolution	Low crosslinking efficiency	Licatalosi et al. (2008)
iCLIP	HITS-CLIP with modifications whereby a 5′ adapter and random barcode is attached to cDNA molecules after reverse transcription; the former modification allows for circularization of the cDNA	Introduction of a random barcode enables identification and quantification of unique cDNA products; cDNA circularization allows for the capture and sequencing of truncated cDNAs usually lost with standard CLIP, revealing crosslinking sites at nucleotide resolution	Low crosslinking efficiency	König et al. (2010)
PAR-CLIP	Photoreactive ribonucleoside analogs (e.g., 4SU or 6-SG) are incorporated into mRNA; nuclease digestion and IP of RBP of interest isolates RBP-protected mRNA fragments	Use of 4SU or 6-SG increases crosslinking efficiency; exact crosslinking sites are revealed after sequencing by T to C transitions in the cDNA prepared from RBP-bound mRNA	Some RBPs may not be amenable to PAR-CLIP	Hafner et al. (2010), Castello et al. (2012)
iPAR-CLIP	PAR-CLIP method applied to C. elegans exposed to 4SU	First demonstration of CLIP in a non-cell line system; allows for physiologically relevant, context-dependent studies of protein-RNA interactions in C. elegans	Technique yet to be applied to other in vivo models	Jungkamp et al. (2011)