**3.5. RNA immunoprecipitation sequencing (RIP-seq)**

including identification and analysis of motifs, differential analysis and association with expression data for deep understanding of bacterial regulon. This is shown in Figure 3 [36].

As whole-genome transcription profiling cannot reveal whether the influence of the transcrip‐ tion factors (TF) on RNA levels is direct or indirect, this requires identification of transcription factors binding within the appropriate promoter region. ChIP-seq provides information about where the TF are bound. Thus, by integrating ChIP methods and transcription profiling, it is possible to identify all direct regulatory targets of a TF for a given condition. For example, work carried out by Stringer et al. (2014) on the *araC* gene of *Escherichia coli* and *Salmonella enterica* has identified direct regulatory targets of AraC, including five novel target genes: *ytfQ*, *ydeN*, *ydeM*, *ygeA* and *polB* [42]. Although ChIP-seq has been used only in moderation to study bacterial systems in a few bacterial species, such as *Vibrio harveyi*, *V. cholerae*, *Rhodobacter sphaeroides*, *Mycobacterium tuberculosis*, *S. enterica* and *Caulobacter crescentus* [36, 37, 43–45], it is

used to identify novel regulatory interactions, even for well-studied proteins [46, 47].

ChIP-seq, in combination with RNA-seq, could be an efficient tool to get detailed information about bacterial transcription regulation and how bacteria respond to different external

**Figure 3.** ChIP-seq sample preparation and analysis. Adapted from [36].

214 Next Generation Sequencing - Advances, Applications and Challenges

conditions.

RNA immunoprecipitation (RIP) is the study of intracellular RNA and protein binding; it is a tool for understanding the dynamic process of post-transcriptional regulatory networks. With this technique, an antibody is used against a protein of interest to recover the RNA species bound to the protein. Since the sequence information of the RNA species bound to a specific protein is often desired, an approach combining RNA immunoprecipitation with sequencing technology (RIP-seq) was created [48]. The main challenge of RIP-seq is the cross-linking step, which is relatively inefficient and only a small amount of RNA is available to construct the library [48, 49]. After that step, treatment with endonuclease elucidates the specific binding sites within the RNA, as they will be protected from digestion. This is followed by purification of the RNA–protein complexes using electrophoresis and high-throughput sequencing [48, 50]. Finally, the data obtained from the sequencer are analyzed using bioinformatics tools. The first study using the RIP-seq-based technique was carried out on *Salmonella* by Sittka et al. (2008) [51]. They used the RNA-binding property of the Hfq protein in their analysis and, as a result, many new sRNA were discovered [52]. Thus, RIP-Seq could be an efficient tool for the identification of bacterial non-coding RNAs.
