Table 1. Characteristics of high throughput RNA structure probing methods.

These techniques use a series of structure probing, extraction, processing, next generation sequencing library preparation, sequencing and bioinformatic data analysis steps (Figure 3) to characterize RNA structures in high throughput and in some cases transcriptome-wide. Detailed differences are included below.

Name	Modifying reagent or enzyme	Probing, Extraction and Processing before Sequencing Library Preparation	Organism(s) studied	Current Analyzing Software	Reference(s)
PARS (parallel analysis of RNA structure)	V1 (dsRNase) & S1 (ssRNase)	RNA extraction, equilibrium refolding, enzyme treatment, RNA fragmentation & linker ligation	Saccharomyces cerevisiae	Bowtie2, custom scripts	Kertesz, 2010, Nature [30]
FragSeq	P1 (ssRNase)	RNA extraction, equilibrium refolding, enzyme treatment, fragmentation & linker ligation	Mus musculus	FragSeq algorithm	Underwood, 2010, Nat. Meth. [29]
PIP-Seq	RNase ONE (ssRNase) & V1 (dsRNase)	crosslinking, cell lysis, enzyme treatment, crosslink reversal, RNA extraction	Homo sapiens, Arabid opsis thaliana	Tophat, CSAR	Silverman, 2014, Genome Biol. [31]
DMS-Seq	DMS	in-cell DMS modification, RNA extraction, fragmentation & linker ligation	Saccharomyces cerevisiae	SOAP	Rouskin, 2014, Nature [35]
structure-seq	DMS	in-cell DMS modification, RNA extraction, random priming	Arabidopsis thaliana	StructureF old (as part of Galaxy suite)	Ding, Nature, 2014 [36]
Mod-Seq	DMS	in-cell DMS modification, RNA extraction, fragmentation & linker ligation	Saccharomyces cerevisiae	Mod-seeker	Talkish, RNA, 2014 [37]
CIRS-Seq	DMS, CMCT	RNA extraction, DMS or CMCT modification, random priming	Mus musculus	custom scripts	Incarnato, Genome Biol, 2014 [38]
icSHAPE (in vivo click selective 2′-hydroxyl acylation and profiling experiment)	NAI-N3 (2-methylnico tinic acid imidazolid e)	in-cell NAI-N3 modification, RNA extraction, fragmentation, biotin click & purification	Mus musculus	Bowtie2, custom scripts	Spitale, Nature, 2015 [39]
RPL (RNA proximity ligation)	Endogeno us RNases	spheroplast,endogenous RNase cleavage, RNA cross-strand ligation, fragmentation & ligation	Saccharomyces cerevisiae	STAR aligner, custom scripts	Ramani, Nat. Biotech., 2015 [41]
SHAPE-MaP (selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling)	1M7 (1-methyl-nitroisatoic anhydride)	in vitro synthesis or viral purification, equilibrium folding, in-cell 1M7 modification, RNA extraction and fragmentation, random/targeted priming & ligation	Hepatitis C & HIV, Mus musculus	ShapeMa pper	Mauger, PNAS, 2015 & Lavender, Plos Comput. Biol., 2015 [40,58,59]
In-cell SHAPE-Seq	1M7	In-cell 1M7 modification, RNA extraction, specific priming	Eschericia coli (natural and synthetic sRNAs, riboswitches and RNase P)	Spats	Watters, NAR 2016 [47]