Computational protocol: The requirement of IRE1 and XBP1 in resolving physiological stress during Drosophila development

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Protocol publication

[…] RNA was isolated from second-instar larvae (48 h old) of Xbp1ex79−/− and Xbp1+/+ genotypes. As an Xbp1+/+ control, a precise excision line from Xbp1CB02061 was used. The latter is the line that was also used to derive an imprecise excision allele Xbp1ex79 (). Two independent extractions of RNAs from each genotype were purified through Qiagen RNeasy columns. RNAseq libraries were prepared using the Illumina TruSeq Stranded Total RNA library prep, with Ribozero Gold, starting from 500 ng of DNase I-treated total RNA, following the manufacturer's protocol, with the exception that 13 cycles of PCR were performed to amplify the libraries, to keep the duplication rate lower than with the recommended 15 cycles. The amplified libraries were purified using AMPure beads, quantified by Qubit and qPCR, and visualized in an Agilent Bioanalyzer. The libraries were pooled equimolarly, and loaded at 8 pM, on a high output HiSeq 2500 flow cell, as paired 50 nucleotide reads for sequencing at the NYU Langone Genome Technology Center. The sequencing reads were aligned to the dm6 reference genome using the Tophat/2.1.1 sequencing analysis package, and HTC count 0.6.1 was used to generate raw gene counts. The package DESeq2 was used for differential gene analysis. The full results have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE99676. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for functional annotation (http://david.abcc.ncifcrf.gov/) of genes whose expression changed significantly in Xbp1 mutants (P<0.05). Network analysis for the selected genes was performed using The Search Tool of the Retrieval of Interacting Genes (STRING) databases (http://string-db.org). Analyzed network and expression level of individual genes were visualized by Cytoscape software (ver 3.4.0). […]

Pipeline specifications

Software tools TopHat, DESeq2
Databases GEO
Application RNA-seq analysis
Organisms Drosophila melanogaster, Homo sapiens, Caenorhabditis elegans