Computational protocol: Decreased expression of lethal giant larvae causes ovarian follicle cell outgrowth in the Drosophila Scutoid mutant

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Protocol publication

[…] Thirty pairs of ovaries were collected and dissected from 1-week old female flies that were cultured at 25°C. The genotypes of the flies were c587-GAL4, UAS-flp/+; ubi-gfp FRT40A/FRT40 or c587-GAL4,UAS-flp/+;ubi-gfp FRT40A/ScoFRT40A. Total RNA were extracted by Trizol reagent (Invitrogen, USA) according to the instructions. RNA was quantified at by absorbance at 260 nm using a ND-1000 spectrophotometer (Nanodrop Techonology, USA) and quality was assessed using a Bioanalyzer 2100 (Agilent Technology, USA) with a RNA 6000 labchip kit (Agilent Technologies, USA). All RNAseq procedures were carried out according to the manufacturer’s protocol from Illumina. Library construction for all samples was accomplished with Agilent’s SureSelect Strand Specific RNA library Preparation Kit for 75SE (Single-End or Paired-End) sequencing on Solexa platform. The sequence was directly determined using sequencing-by-synthesis technology with a TruSeq SBS kit. Raw sequences were obtained from the Illumina Pipeline software bcl2fastq v2.0 and expected to generate 12.5M (million reads) per sample. The sequences were then filtered to obtain qualified reads. Trimmomatic software was implemented to trim or remove the reads according to the quality score. The gene expression level was calculated as FPKM (Fragment Per Kilobase of transcript per Million mapped reads). For differential expression analysis, CummeRbund was used to perform statistical analysis of gene expression profiles. The reference gene annotations were retrieved from Flybase. Data was deposited in the NCBI GEO under the accession number GSE43506. […]

Pipeline specifications

Software tools BCL2FASTQ Conversion Software, Trimmomatic, CummeRbund
Databases FlyBase
Applications RNA-seq analysis, Transcriptome data visualization
Organisms Caenorhabditis elegans, Drosophila melanogaster
Chemicals Zinc