Computational protocol: Transcriptome Profiling Identifies Multiplexin as a Target of SAGA Deubiquitinase Activity in Glia Required for Precise Axon Guidance During Drosophila Visual Development

Similar protocols

Protocol publication

[…] Four biological replicates were analyzed for each of the following genotypes: wild type, nonstop, and sgf11. Quality trimming was performed on paired-end reads for all 12 samples using Trimmomatic (v0.32) () to remove bases with Phred33 < 30, resulting in properly paired reads of at least 50 bases. Quality trimmed reads were mapped against the bowtie-2 (v2.2.4) () indexed D. melanogaster genome (Drosophila_melanogaster.BDGP5.78) using Tophat (v2.0.13) (). The raw counts matrix was generated by Htseq-count (v0.6.1) applying no strand-specific assay, union mode, and default parameters (). Differential expression analysis was performed on genes with greater than one count per million (CPM) in at least four of the 12 samples. Differentially expressed genes were detected in each mutant genotype relative to the wild-type genotype using edgeR () using a False Discovery Rate (FDR) of less than 0.01. The distance matrix and scatter plots were generated using Bioconductor packages of DESeq2 () and edgeR (), respectively, in R (v3.1.2). Gene Ontology (GO) term enrichment analysis was performed using a Fisher’s exact test and significantly enriched GO terms were defined as those with a FDR < 0.001. Actively transcribed genes were defined as fragments per kilobase of transcript per million mapped reads (FPKM) of greater than one in wild-type glia. The GO term analysis used the Bioconductor Drosophila genome annotation package 3.1.2 with GO data from March 17, 2015. GO terms with less than eight or more than 250 genes were removed, as were gene annotations with no supporting data.The RNA-seq data for the central nervous system of OregonR larvae were obtained from the modENCODE project: Dm Tissue Expression RNA-seq third instar larvae central nervous system sequences (ModENCODE_4257). Two biological replicates for larval central nervous system RNA-seq data were mapped against the bowtie-2 (v2.2.4) () indexed D. melanogaster genome (Drosophila_melanogaster.BDGP5.78) using Tophat (v2.0.13) (). A raw counts matrix was generated by Htseq-count (v0.6.1) applying no strand-specific assay, union mode, and default parameters (). A count matrix for the central nervous system and the wild-type glia nuclei was assembled, and edgeR was used to normalize libraries and determine the FPKM values for genes that had greater than one CPM in two or more of the six samples. [...] Exonic primers flanking intron regions of target genes were designed for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis using Primer3. qRT-PCR analysis was performed on cDNA as previously described (). […]

Pipeline specifications

Software tools Trimmomatic, Bowtie, TopHat, HTSeq, edgeR, DESeq2, Primer3
Databases BDGP
Applications RNA-seq analysis, qPCR
Organisms Drosophila melanogaster, Homo sapiens
Diseases Retinal Degeneration, Machado-Joseph Disease