**4. RNA extraction**

**2.3. Colonization of GFP-tagged bacterium in the gut of** *S. littoralis*

bacterial cells.

82 Metagenomics for Gut Microbes

**3. Fluorescence-activated cell sorting**

tive data of a particular kind of cell can be assessed.

The fluorescent reporter *E. mundtii* has been integrated into the gut microbiome across all developmental stages of *S. littoralis* [14], indicating its symbiotic relationship with the insect host. Microorganisms have the ability to face environmental stresses, particularly those within the gastrointestinal environment. Constructing the fluorescent reporter *E. mundtii*, we explored the mechanisms these bacteria use to adapt to stress; we recovered the reporter bacteria from the gut of *S. littoralis* using the state-of-the-art technology of flow cytometry. The dominance and persistence of *E. mundtii* in the gut motivates us to look deeper into their gene expression system. Therefore, it is important to unravel the mechanisms used by microorganisms living within the gastrointestinal environment. Construction of the fluorescent reporter *E. mundtii* is one of the strategies to find out those mechanisms, since it has been possible to recover the reporter bacteria from the gut of the very same insects using the *state-of-the-art* technology of flow cytometry. Fluorescence-activated cell sorting (FACS) enabled us to pick out the GFP-tagged reporter *E. mundtii* from a mixture of insect and other

Flow cytometry separates cells based on their intrinsic physical and chemical characteristics, integrating electronics, fluidics and optics. The sample, from which the cells of interest are to be sorted, is passed through a flow cell. The sheath fluid escorts the cells down the channel, where they encounter a laser beam. Light beams of specific frequencies and wavelength are emitted. Detectors measure the forward scatter (FSC) and the side scatter (SSC) based on cell size and granularity. FSC and SSC are unique for every particle. A combination of the two can differentiate among cell types within a cohort of cells. This way, the qualitative and quantita-

Fluorescence-activated cell sorting is an application of flow cytometry. The cells of interest are fluorescently tagged and sorted by the machine. Here the GFP-tagged fluorescent *E. mundtii* is isolated from a mixture containing insect gut homogenate with other bacteria. The solution is delivered to the flow channel and carried by the sheath fluid. The pressure from the compressor, which is adjustable, forces the solution through a laser beam using hydrodynamic focusing. Then monochromatic beams of high intensity interrogate cells one at a time. Depending on the excitation wavelength of the fluorophore, the laser wavelength is chosen. The scatters are then recorded. The forward scatter (FSC), which refers to light that is refracted by the cell and continues in the same direction, tells us about the size of the cell. In contrast, the side scatter (SSC), which refers to light that is refracted by the cells and travels at right angles to the excitation axis, tells us about the fluorescence and granularity of the cells. The more granular a cell, the more scattered light it produces. Furthermore, each cell enclosed in a droplet is assigned a charge, depending on the extent of the cell's deflection [90]. After passing through an electrical field, the cells are deflected to the collection tubes and the uncharged droplets are directed to the waste. The detector system consists of The GFP-tagged *E. mundtii* are sorted by the flow cytometer and collected in a RNA-protective reagent (RNAlater®). The role of such reagents is twofold: first, they preserve the integrity of RNA, which has a very short half-life, for a few minutes. We need the RNA to be intact and of good quality in order to process it for sequencing. Second, addition of protective reagents minimizes subsequent changes from being introduced when the cells are handled. As soon as the cells are collected in a Falcon tube filled with the protective reagent (RNA Protect or RNA Later), the reagent percolates into the cells and prevents an alteration in the gene expression [91]. Additionally, the entire process is maintained at 4°C, as all metabolic activities slow down at low temperatures. The Falcon tube is centrifuged at a high speed to pellet down the cells, and care is taken not to disturb it while draining the supernatant.

RNA is very sensitive to exogenous and endogenous RNases. The entire extraction procedure is done in an area free of RNase. Moreover, RNase inhibitors are used to clean all equipment, ranging from gloves to microcentrifuge tubes to get rid of RNase. *E. mundtii* is a Gram-positive bacterium with a cell wall containing a thick layer of peptidoglycan and lipoteichoic acid, followed by a single lipid membrane. The cell wall is anchored to the membrane by diacylglycerols. To release the nucleic acid from the cell, it has to be detached from its peptidoglycancontaining cell wall and membrane. Lysozyme is a glycoside hydrolase that hydrolyzes the 1,4-beta linkages between *N*-acetylmuramic acid and *N*-acetyl-D-glucosamine residues of the peptidoglycan. Additionally, guanidium thiocyanate, beta-mercaptoethanol and a detergent called dithiothreitol help in cell lysis and deproteinization. Proteinase K frees the RNA from the bound proteins and endogenous RNase.

Following lysis, the RNA is separated by density gradient centrifugation using phenol, chloroform and isoamyl alcohol, and further precipitation with ethanol. The RNeasy® Mini Kit (Qiagen) based on silica-matrix RNA extraction was used in our work. Several studies have reported on extraction of high quality bacterial RNA using this kit [92–94]. Thus, RNA is obtained from the cells of the sorted *E. mundtii* [95].

The extraction of total RNA from the low number of bacterial cells that remain after sorting by the flow cytometer is challenging. The concentration of RNA was as low as a few picograms to 50 ng. The minimum threshold quantity for a successful RNA library preparation is 100 ng. This is too low an amount to proceed with RNA sequencing. Hence, the total RNA must be amplified before we can use it further.

#### **4.1. Amplification of RNA**

Amplification of RNA is required if the aim is to create an effective transcriptomic profile from a very low starting quantity of RNA. MessageAmp II aRNA amplification kit (Ambion) was used for amplification [96–99]. The principle is based on *in vitro* transcription. The steps are as follows:

Certain amplified transcripts may be misunderstood as duplicates and *vice versa*, which could give a false positive read [105]. In PCR-based amplification procedures, duplicates that can arise from sample handling may have features such as fragmentation, sequencing depth or library complexity; unfortunately, these cannot be distinguished from PCR duplicates. Removing duplicates does not improve the accuracy of quantification or the power; rather, makes it worse [106]. The *Taq* polymerases used for the PCR-based approach are more prone to introduce errors than the RNA polymerases for *in vitro* transcription. Thus, *in vitro* transcription is favored over PCR-based amplification [105], although premature transcription termination can occur in low complexity sequences [107]. Nevertheless, *in vitro* transcription

The Microbiome of *Spodoptera littoralis*: Development, Control and Adaptation to the Insect Host

http://dx.doi.org/10.5772/intechopen.72180

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At this point, we have enough RNA to get a transcriptomic profiling of the bacterial cells done. The transcriptome is the entire set of genes expressed in a type of cell at a particular time point and/or condition. This is in contrast to a genome, which refers to the full complement of genes in a cell-type. Not all genes are constitutively induced. Information about transcripts, or genes expressed, may shed light on the developmental or physiological state of the cell. It also talks about other species of RNA, small RNAs and non-coding RNAs, novel transcripts, the transcriptional start sites, splicing regions, post-transcriptional modifications, and 3′ and 5′ ends. Another purpose of transcriptomic profiling is to quantify the expressed genes. One can judge the extent of regulation of a particular gene in the given conditions. As compared to one situation, when cells behave differently in another, one can now say which

In this chapter, our aim has been to investigate the survival and adaptation strategies of *E. mundtii* living inside the gut of *S. littoralis* as compared to in the laboratory. This unraveling has been done by cataloging the genes of *E. mundtii* which are differently regulated and which make it as

Transcriptome sequencing has improved dramatically over the past few years, starting with EST-based Sanger sequencing. The early method was mainly useful with the most abundant transcripts, whereas subsequent next-generation sequencing has been successfully carried out on all transcripts with sensitivity and accuracy even allowing the identification of low expressed genes. The situation has ameliorated with the advent of deep sequencing, which can increase the average number of times a nucleotide is sequenced. The deeper the sequencing is, the better the probability of detecting the less abundant transcripts. Next-generation sequencing has several hierarchies of its own. These days, RNA-seq is more widely used than the microarrays. The former gives us a base-pair level of resolution. Whereas microarrays can be used only when the reference genome sequence is available, RNA-seq can build the transcriptome *de novo.* Also, background noise is taken better care of in the case of RNA-seq. These

is an efficient method to follow when the starting quantity is limited [107].

genes are differentially regulated to bring about the same.

one of the dominant bacterial species in the gut.

**5. Transcriptomics**

**5.1. RNASeq**

#### *4.1.1. Polyadenylation of RNA*

Bacterial RNA is devoid of a poly (A) tail. The *E. coli* poly (A) polymerase enables a poly (A) tail to be added at the ends of RNA. This stretch is required for cDNA synthesis.

#### *4.1.2. Synthesis of first-strand cDNA*

Primers against the poly (A) stretch are used to synthesize the first strand of cDNA by reverse transcription. The primers are anchored with a bacteriophage promoter sequence: T7 oligo(dT) sequence, T3 or SP6. dNTPs are added to the reaction mix.

#### *4.1.3. Synthesis of second-strand cDNA*

RNaseH is used to degrade the RNA from the RNA-cDNA pair; DNA polymerase is required to synthesize the second strand of cDNA. The result is a double-stranded cDNA fragment with a T7 promoter sequence.

#### *4.1.4. Purification of cDNA*

cDNA is cleaned by removing the fragmented RNA, enzymes and salts, all of which could hinder *in vitro* transcription.

#### *4.1.5. In Vitro transcription*

Multiple copies of antisense RNA are generated using DNA-dependent RNA polymerase. Linear amplification is employed for this. Depending on the bacteriophage promoter sequence attached to the cDNA, a polymerase is selected. Promoter-specific dNTPs are added to the reaction mix. 37°C is optimum for this reaction. The reaction time depends on the extent to which one wants to amplify the RNA.

#### *4.1.6. Purification of amplified RNA*

The residual enzymes, salts and unincorporated dNTPs must be removed from the final product [100–104].

At this point, the RNA has been amplified several fold: 1–2 ug.

RNA amplification procedures have a drawback. When the concentration of RNA is brought to a point at which successful transcriptomic profiling is possible, certain biases are unavoidably introduced.

Certain amplified transcripts may be misunderstood as duplicates and *vice versa*, which could give a false positive read [105]. In PCR-based amplification procedures, duplicates that can arise from sample handling may have features such as fragmentation, sequencing depth or library complexity; unfortunately, these cannot be distinguished from PCR duplicates. Removing duplicates does not improve the accuracy of quantification or the power; rather, makes it worse [106]. The *Taq* polymerases used for the PCR-based approach are more prone to introduce errors than the RNA polymerases for *in vitro* transcription. Thus, *in vitro* transcription is favored over PCR-based amplification [105], although premature transcription termination can occur in low complexity sequences [107]. Nevertheless, *in vitro* transcription is an efficient method to follow when the starting quantity is limited [107].
