Computational protocol: Multimodal stimulus coding by a gustatory sensory neuron in Drosophila larvae

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Protocol publication

[…] For RNAseq experiments the protocol for the preparation of a cell suspension after Egger et al. was slightly modified and applied as followed.For dissection, 5-day old feeding L3 larvae were collected and washed in 70% ethanol followed by PBS (two repetitions). Forty to fifty mouthparts (MPs) were dissected in Schneider's Medium (Gibco), than rinsed in fresh medium and subsequently transferred to a siliconized microcentrifuge tube with 1-ml Rinaldini solution. From here on, experiments were performed under laminar flow in sterile conditions. The MPs were centrifuged for 6 min at 900g at RT. The supernatant was carefully removed and MPs were washed in Rinaldini solution. The Rinaldini solution was replaced by 1 ml collagenase I (0.5 mg ml−1, sterile filtered, Sigma-Aldrich). The digestion was allowed to take place for 40 min at RT and subsequently, the tissue was centrifuged 6 min at 850g at RT and collagenase was replaced by Schneider's medium. After 6 min of centrifugation at 750g at RT all the supernatant was removed and 10 μl of Schneider's medium per MP were added. With a Pipette, set to half the contained volume, the solution was pipetted up and down 100–200 times.The suspension was pipetted on a small petri dish containing Sylgard (Dow Corning) and organs were manually sorted under a fluorescent microscope with a glass capillary (GC100TF-10, Harvard Appartus) and subsequently transferred in 100 μl of Lysis Buffer (From PicoPure RNA Isolation Kit, Arcturus). The RNA extraction was performed as suggested by the Kit protocol. Samples were additionally treated with DNAse (Quiagen, catalog 79254).After RNA extraction, samples were treated exactly as recommended in SMARTer Ultra Low RNA Kit for Illumina sequencing (PT5163-1 (051013)) to transcribe RNA into cDNA and purify the samples.The cDNA volume was reduced from 10 to 1 μl by using speed vac for ∼9 min. Libaries were prepared using the TruSeq DNA Sample Preparation Guide without gelsize selection, quality was checked after covaris shearing by Bioanalyzer 100 and Qubit. Samples were paired-end sequenced (2 × 100b) on an Illumina HiSeq2500 instrument.The quality of the sequence reads was assessed using fastQC version 0.10.1 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The reads were mapped to the Drosophila melanogaster genome assembly (dm3) using the spliced alignment tool TopHat2 (v2.0.6) with default parameters. The number of reads that map to annotated genes (release 5.12) was counted with HTSeq-count (ref. ). To test for significant differences of gene expression levels between DOG and TOG the R/bioconductor-package DESeq2 (ref. ) was applied. The P values were corrected for multiple testing following and threshold of 0.05 was applied. The R/bioconductor-package DESeq2 (ref. ) was used to perform a differential gene expression analysis between DOG and TOG. A size-factor normalization was employed to account for differences in the sequencing depth of the samples. A generalized linear model was fitted to the data, allowing the error distribution to follow a negative binomial function. The significance of the observed differences was determined by means of a Wald test. The P values were corrected for multiple testing following and a threshold of 0.05 was applied. Expression of sensory receptor genes within the TOG and DOG are summarized in . […]

Pipeline specifications

Software tools FastQC, TopHat, HTSeq, DESeq2
Application RNA-seq analysis
Organisms Drosophila melanogaster
Chemicals Calcium, Sucrose