Table 1. Next-Generation Sequencing Tools to Analyze Global Changes in Chromatin Landscape, AS, RNA Structure, and Targets of RBPs
MethodDescription
A. AS
 RNA-seqcDNA generated from polyadenylated mRNA is fragmented and sequenced by next-generation sequencing. Alignment of reads to the genome allows quantitative analysis of gene expression, AS, and relative quantification of mRNA isoforms (Wang et al., 2008).This tool is also used to sequence other types of RNA.
B. Chromatin landscape
 Bisulphite sequencingGenomic DNA treated with bisulphite, which converts unmethylated cytosines to uracils, is amplified and sequenced using high-throughput methods. In the sequenced reads, unmethylated cytosines appear as thymines and methylated cytosines appear as cytosines (Cokus et al., 2008). Provides a genome-wide cytosine methylation map at single-base-pair resolution.
 DNAseI-seqNuclei are treated with DNase I, and short sequence tags are obtained, size selected, and sequenced using next-generation sequencing. The short reads are then mapped to the genome and clustered computationally to identify DNAase I hypersensitive sites (Boyle et al., 2008; Hesselberth et al., 2009). Allows genome-wide mapping of open chromatin, which is hypersensitive to DNAase I.
 Micrococcal-nuclease sequencingIsolated chromatin is digested with MNase to remove DNA that is not wrapped around nucleosomes, and DNA from the nucleosomes is extracted and subjected to deep sequencing. Alignment of reads to the genome provides global nucleosome occupancy (Barski et al., 2007; Chodavarapu et al., 2010).
 Chromatin-immunoprecipitation sequencingChromatin is immunoprecipitated with an antibody specific to a histone modification or a DNA binding protein, and the DNA from the precipitate is extracted, amplified, sequenced on a next-generation sequencing platform, and mapped to the genome (Furey, 2012). Used to analyze genome-wide protein–DNA interactions and histone modifications.
C. Pre-mRNA/mRNA structure*
 Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) sequencingRNA is treated with a SHAPE reagent (e.g., 1-methyl-7-nitroisatoic anhydride, IM) that forms 2’-O-adducts with unpaired nucleotides and blocks the extension of cDNA during reverse transcription, resulting in termination of synthesis. All generated fragments are sequenced and mapped to RNA to determine its structure (Lucks et al., 2011). Allows simultaneous measurement of secondary and tertiary structures of large numbers of RNAs in vitro.
 PARS sequencingIsolated mRNAs are renatured in vitro, treated with structure-specific enzymes (RNAse V1, which preferentially cleaves phosphodiester bonds 3′ double-stranded RNA or RNAase S1 that cleaves single-stranded RNA) in parallel, and the resulting RNA fragments are sequenced using next-generation sequencing (Kertesz et al., 2010; Wan et al., 2013). Allows in vitro profiling of the secondary structures of RNA at single-nucleotide resolution globally.
 Double-stranded RNA sequencingRNAs are treated with single-strand- specific RNAases (RNAse ONE) to remove single-stranded regions and select for double-stranded RNA, which is then converted into cDNA for deep sequencing (Zheng et al., 2010). Reads are then used to obtain structural information.
 Single-stranded RNA sequencingRNAs are treated with double-strand–specific RNAases (RNAseV1) to remove double-stranded regions in RNA and select for single-stranded regions of RNA. These are then converted into cDNA for deep sequencing. These reads together with double-stranded RNA sequencing are then used to identify structural elements in RNA (Li et al., 2012).
 Fragmentation sequencingRNA mixture is treated with nuclease P1, which specifically cleaves single-stranded RNA followed by RNA-seq to deduce transcriptome-wide RNA structure (Underwood et al., 2010).
D. Targets of RBPs
 RIP sequencingRNAs bound to a specific RBP are immunoprecipitated, and the RNA from IP is converted into cDNA and sequenced using a high-throughput sequencing platform (Zhao et al., 2010). The reads are then used to identify direct and indirect targets of that RBP.
 HITS-CLIPRBP–RNA interactions are first covalently cross-linked by exposing cells/tissues to UV (254 nm). An RBP-specific antibody is used to precipitate RNA bound to RBP, which is then treated with RNAse and used for RNA-seq. iCLIP (individual-nucleotide-resolution UV cross-linking and immunoprecipitation) is a variation of HITS-CLIP that exploits premature termination of reverse transcriptase at the cross-link site, thereby providing better resolution of binding sites (König et al., 2010). Allows genome-wide mapping of direct binding sites of an RBP on RNA (Licatalosi et al., 2008).
 Individual-nucleotide-resolution UV-cross-linking and affinity purificationDouble-tagged RBP is expressed in cells. Following UV cross-linking and RNAase digestion, the RBP-RNA complex is purified by a two-step affinity purification. RNA from these complexes is then deep sequenced (Wang et al., 2010b). Allows purification of RBP-RNA complexes.
 Photoactivatable-ribonucleoside-enhanced CLIPSimilar to HITS-CLIP except that RNA is labeled with 4-thiouridine and cross-linking is done using UV light of 365 nm (Hafner et al., 2010b). This allows efficient cross-linking of RBPs with RNA, and identification of nucleotides involved in binding can be identified as the cross-linked regions show T-to-C mutations in the reads. A similar approach (photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation) is also used to identify all RBPs bound to mRNAs and to create binding maps of multiple RBPs. In this case, mRNAs cross-linked with RBPs are isolated using oligo(dT) affinity purification and digested with RNAse followed by identification of proteins by mass spectrometry and RNA by RNA-seq (Baltz et al., 2012).
  • * These in vitro methods are based on high-throughput RNA-seq and can be applied to all types of RNAs.