Table 1. Comparative Characteristics of the Protocols Discussed in the Review.

Parameters	Protocol
	RT-Seq	NO-Seq	AlkAniline-Seq	HydraPsiSeq
Detected modification	m1A, m1G, m22G, m3U, m3C, m66A, other residues modified at the WC face	m6A	m7G, m3C, D, ho5C/ho5U, RNA abasic sites	Ψ, m5U, 5-modified U*34, k2C (positive cleavage signal), m7G (positive cleavage signal)
Nature of the signal	RT stop + misincorporation/jump signature	Resistance of m6A to deamination compared to unmodified A	Cleavage at the abasic RNA site resulting from decomposition of the modified residue	Resistance of Ψ, m5U, 5-modified U*34 residues to hydrazine cleavage, over cleaved U residues
Detection type	positive	negative	positive	negative
Input RNA required	>100 ng	∼ 500 ng	>50 ng	>150 ng
Molar sensitivity	average	low	very high	average
Quantification	Possible, but complicated	Precise	Possible, but calibration is not linear	Precise
Reagents used	None (different RT enzymes recommended)	NaNO2/H+	Alkaline hydrolysis/Aniline	Hydrazine/Aniline
Library preparation step	Custom protocol	Custom protocol	NEB Small RNA kit	NEB Small RNA kit
Required sequencing depths	Coverage >100 reads/position, better >500	Coverage >50 reads/position	>50 reads/RNA position	>1000 reads/RNA position
Recommended size of ≪ limited transcriptome ≫	10 000 bases rRNA/tRNA size	100 bases Custom amplicons	10 000 000 bases Transcriptome-wide	10 000 bases rRNA/tRNA size
Bioinformatics analysis	straightforward	Complicated (custom alignment algorithm)	straightforward	Intermediate (normalization and score calculations)