TITLE: Total RNA extraction from transgenic flies misexpressing foreign genes to perform Next generation RNA sequencing
AUTHORS: Abijeet Mehta, Agustin Luz-Madrigal, Jian-Liang Li, Panagiotis A. Tsonis, Amit Singh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Due to absence of transgenic approaches in </span><span style = "font-style:italic;">Notopthalmus Viridescens (newt)</span><span>, and conservation of genetic machinery across species, we generated transgenic </span><span style = "font-style:italic;">Drosophila melanogaster</span><span> to misexpress unique genes from newt. Novel newt genes cloned, and inserted at attP site in Drosophila  were misexpressed ubiquitously using tubulin Gal-4. Sample (total RNA) for RNA sequencing was collected at 3</span><span style = "vertical-align:super;">rd</span><span> instar larval stage during which major developmental events takes place in </span><span style = "font-style:italic;">Drosophila</span><span>. Total RNA was extracted, and purified using RNA clean and Concentrator</span><span style = "vertical-align:super;">TM</span><span>. RNA quality was quantitated  by calculating absorbance at 260 nm (A260) and 280 nm (A280) wavelengths using Nanodrop 2000 spectrophotometer.  Good quality samples had A260/ A280 ratio greater than 2 and a peak at 260 nm. Our results show that following this protocol high quality of RNA was obtained. These high quality RNA samples were used for downstream processes e.g.  Next generation RNA sequencing. Of the total 36,099 transcripts in </span><span style = "font-style:italic;">Drosophila, </span><span>34,967 transcripts were detected, and 2775 transcripts were significantly regulated by misexpressing foreign gene (Unique gene from newt) in </span><span style = "font-style:italic;">Drosophila . </span><span>Genes involved in the developmental process, cell cycle, apoptosis, and immune response are among those that are highly enriched. Wingless/Wnt was one of the important evolutionarily conserved pathway that was differentially regulated.</span></div><style>
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?. Sample collection and StorageTransgenic Drosophila melanogaster were generated by genetivision (https://www.genetivision.com/). Briefly, transgenic flies were generated using microinjection-based ϕC31 integrase mRNA-mediated method. A cloned candidate gene using pUAST-attB plasmid containing both a transgene and donor sequence (attB) is coinjected along with ϕC31 integrase mRNA into attP-containing recipient embryos, resulting in the site-specific insertion of the transgene (Fish et al., 2007; Groth et al., 2004). Following this procedure 5 independent transgenic flies were generated with insertion of newt gene at Chromosome III.Foreign gene of interest was misexpressed ubiquitously using tubulin Gal-4. The third instar larvae were selected and were stored in RNAlater (Thermo Fisher, Cat. No. # AM7024) solution. The samples were then stored at 4°C. For long term storage we isolated larvae into empty RNase free tubes and store them at -70°C.
?. RNA isolation and PurificationThe tubes were briefly centrifuged and RNAlater solution was completely removed. We added 500 μl of TRIzol Reagent (Thermo Fisher, Cat. No. # 15596926), which is used to isolate good quality RNA from tissue samples. TRIzol is a monophasic solution, primarily consisting of phenol and guanidine isothiocyanate along with other proprietary components, which disrupt samples, stabilize nucleic acids, and are compatible with downstream analysis . Tissue was homogenized in TRIzol using a 150 handheld homogenizer Motor (Fisher scientific, Cat. No. # 15-340-167). The solution was incubated for 30 minutes in ice. Centrifuged for 10 minutes at 4°C and supernatant was transferred to clean tube. Chloroform (Sigma Catalog #: 319988/W205702)was added and homogenate was allowed to separate into a clear upper aqueous layer. 200 μl of the aqueous phase was transferred to RNA clean and ConcentratorTM (Zymo research, Cat. No. R1080) columns, and the recommended protocol was followed, i.e., solution was passed through the RNA binding buffer, which binds RNA to the desired columns. Then RNA wash buffer was added to remove all the impurities from the column. Finally RNA was eluted in 20 μl of molecular grade water (DNase/RNase free) and collected in a separate tube. The molecular grade water serves as the elution buffer. It releases the RNA from the column.
?. RNA quantitation, and StorageRNA quantitation is an important and necessary step prior to most RNA analysis methods. Quality of RNA as well as concentration of RNA was determined by calculating absorbance at 260 nm (A260) and 280 nm (A280) wavelengths using Nanodrop 2000 spectrophotometer (Thermo Scientific). Good quality samples had A260/ A280 ratio greater than 2 and a peak at 260 nm. High quality total RNA was shipped in Dry ice to our collaborator for Next generation RNA sequencing.
?. RNA Sequencing and AnalysisIllumina reads were mapped to the Drosophilagenome dm6 using TopHat splice-aware aligner (Kim et al., 2013). Expectation-Maximization (EM) approach was used to estimate transcript abundance (Jiang and Wong, 2009). Reads per kilobase per million mapped reads (RPKM) approach was applied for within sample normalization (Mortazavi et al., 2008). Between sample normalization and differentially expressed test were performed by BioConductor DESeq package (v 1.20) which allows analysis of non-replicate experiments (Anders and Huber, 2010). The significant criteria were the detected transcript in at least one sample (RPKM>1), fold change over 2 and adjusted p-value less than 0.05. Enriched gene ontology (GO) terms were identified using gene ontology enrichment analysis and visualization tool (GOrilla) (Eden et al., 2009). Single ranked list of gene was chosen as a running mode criteria, and for enriched GO terms searched P-value threshold was equal to 10-3. Using reduce + visualize gene ontology (REVIGO) tool  long lists of gene ontology terms obtained by running Gorilla were summarized by removing redundant GO terms (Supek et al., 2011). The remaining terms were visualized as tree map/ bar graph. We also used protein analysis through evolutionary relationships (PANTHER) 14.1 version as a tool to select set of enriched gene ontology terms for classifications by molecular function, cellular component, and Protein class  (Mi et al., 2019).  
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