Table 1. List of methods for analysis of translation in time and space.

	Method	Advantages	Disadvantages	Ref.
Next-generation sequencing	Polysome profiling	Reproducible; quantitative; high depth of analysis; gives an instantaneous snapshot of the translatome (high temporal resolution).	Contamination by co-sedimented RNPs can be an issue; does not reveal the exact ORF sites in an mRNA; more association of an mRNA to ribosomes may not always mean more translation.	16
	Ribo-seq	High depth of analysis; single-nucleotide resolution; allows de novo ORF detection; highly quantitative; gives an instantaneous snapshot of the translatome (high temporal resolution).	Costly and time consuming; requires a large amount of starting material; more association of an mRNA to ribosomes may not always mean more translation.	15 and 20
	TRAP-seq	Similar to ribo-seq but can be used for cell-specific in vivo analysis of translation.	Similar to ribo-seq, but requires more starting material.	33 and 34
	Proximity-specific ribo-seq	Similar to ribo-seq but can reveal subcellularly localized translation.	Similar to TRAP-seq, but requires even more starting material as only a fraction of total cellular ribosomes are labeled and purified.	36 and 37
Proteomics	p-SILAC	Quantitative; measures nascent proteins; allows analyses from small sample sizes and subcellular compartments.	Low depth; limited temporal resolution due to the need for incorporation of pulsed amino acids into cellular proteins; cannot be readily used in vivo.	14, 39 and 41
	BONCAT	Measures nascent proteins; higher depth than p-SILAC due to enrichment of nascent proteins.	Limited temporal resolution due to the need for incorporation of pulsed amino acids into cellular proteins; cannot be readily used in vivo without utilizing engineered amino acyl-tRNA synthetases; semi-quantitative.	46
	SORT	Similar to BONCAT but can be used for cell-specific in vivo analysis of translation.	Generation of animal models costly and time consuming. Limited temporal resolution due to the need for incorporation of pulsed amino acids into cellular proteins; semi-quantitative.	54
	QuaNCAT	Quantitative like p-SILAC, but at higher depths due to enrichment of nascent proteins; improved temporal resolution in comparison to BONCAT and p-SILAC; measures nascent proteins.	Improved, but still limited temporal resolution due to the need for amino acid pulsing; cannot be readily used in vivo.	56
	HILAQ	Quantitative like p-SILAC, but at higher depths; experimental workflow much simpler that QuaNCAT; improved depth and temporal resolution in comparison to QuaNCAT.	Improved, but still limited temporal resolution due to the need for amino acid pulsing; cannot be readily used in vivo.	59
	PUNCH-P	High depths of analysis; gives an instantaneous snapshot of the translatome (high temporal resolution); measures nascent proteins.	Time consuming; requires prior lysis and purification of translating ribosomes, thus losing any spatial regulatory influences on translation; requires a large amount of starting material; semi-quantitative.	63
	OPP capture	Improved temporal resolution compared to p-SILAC and BONCAT due to rapid OPP incorporation into cellular proteins; measures nascent proteins; can be used for cell-specific in vivo assessment of translation (PhAc-OPP).	Semi-quantitative (as of now).	64 and 65
Live cell imaging	TRICK	Allows live monitoring the first round of translation; single molecule sensitivity; can potentially be used in vivo.	Not high throughput; low signal to noise ratio; cannot be used for assessment of translation rates, but only visualizing the pioneer round of translation.	67
	NCT/SINAPS	Allows continuous monitoring of translation dynamics in live cells over time scales of hours; single molecule sensitivity; reveals translation heterogeneity; can potentially be used in vivo.	Not high-throughput; background fluorescent accumulation over time can be an issue.	69–72