Computational protocol: X Chromosome and Autosome Dosage Responses in Drosophila melanogaster Heads

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Protocol publication

[…] We used congenic Drosophila strains from the European Drosophila deletion collection (DrosDel) project (; ) to assay expression changes due to gene dosage. In addition to the DrosDel lines, we examined expression in the w1118 DrosDel parental line and used a Dp(1;Y)BS; cn1 tra2B bw1/CyO and Df(2R)trix/CyO stocks for sex-transforming flies (Bloomington Drosophila Stock Center, Bloomington, IN). We also used OreR (gift from Elissa Lei, Laboratory of Cellular and Developmental Biology, NIDDK, NIH, Bethesda, Maryland). Flies were grown at 25° on San Diego Stock Center cornmeal media. We collected 30 5-d-old adult flies for each triplicate of 10 heads, which were dissected on dry ice and stored at −80°.We homogenized frozen heads in 2 ml Axygen 96-well plates (Corning, Life Sciences, Union City, CA) preloaded with 200 µL 1-mm glass beads (Biospec Products Inc., Bartlesville, OK) and 200 µL RLT Buffer (Qiagen, Valencia, CA) and covered with Axygen sealing mats, in a Mini-BeadBeater 96 (Biospec Products Inc., Bartlesville, OK). We extracted total RNA with the RNeasy 96 Kit and QIAvac 96 vacuum manifold (Qiagen, Valencia, CA). We visually inspected RNA quality by gel electrophoresis detected with SYbrGold Nucleic Acid Gel Stain and we used a plate reader to quantify yield with Quant-iT RiboGreen (Life Technologies, Grand Island, NY). We used 100 ng of total RNA and the TruSeq RNA Sample Preparation V2 protocol (Illumina Inc., San Diego, CA) to make RNA-seq libraries from polyA+ RNA. We added 10 pg of pool 78A ERCC spike-in RNAs () to the Elute, Prime, and Fragment Mix in the “Purify and fragment mRNA” step. We checked the library quality with the High-Sensitivity DNA Analysis Kit on a Bioanalyzer (Agilent, Santa Clara, CA) and concentration by Quant-iT PicoGreen (Life Technologies, Grand Island, NY), and performed 76nt single-end sequencing on an Illumina HiSequation 2000 running CASAVA-1.8.2/RTA Control Software (Illumina Inc., San Diego, CA). [...] To measure expression genome-wide, we performed single-end RNA-Seq on poly-A+ RNA extracted from adult female and male heads in biological triplicate. We uniquely mapped reads to FlyBase genome release 5.57 () and spike-in RNAs () that we added to the libraries to empirically determine sampling variance in complex mixtures of RNA species. We used DESeq2 () normalized read counts and cufflinks () fragments per kilobase per million mapped reads (FPKM) to report gene-level steady-state expression.We downloaded GFF3 gene annotation files from NCBI (, extracted CDS and exon features on existing chromosomes (excluding chromosome U and Uextra), and converted to GTF format, which is compatible with all analysis tools we used, using a custom Perl script. Some peculiar gene features were incompatible with analysis tools. We “corrected” the boundaries of 10 CDS, which were beyond that of the parent genes and changed strand information for 9 CDS (11 exons) according to the annotation of the parent genes. We also added 96 ERCC spike-in annotations in the final GTF file. We mapped RNA reads with Tophat v2.0.10 (). We used parameter −g 1 to retain only uniquely mapped reads and provided the GTF format annotation.We measured the gene expression using cufflinks v2.1.1 () in fragments per kilobase of transcript per million mapped reads (FPKM). We measured gene expression using HT-Seq v0.5.4p1 (), which reports in read counts. We normalized read counts using DESeq2 v1.6.3 () in R v3.1.1. We used coefficient of variance for genes, intergenic space, and spike-in RNAs to derive a low expression cut-off value (see below). […]

Pipeline specifications

Software tools BaseSpace, HSC, DESeq2, Cufflinks, TopHat, HTSeq
Databases FlyBase
Application RNA-seq analysis
Organisms Drosophila melanogaster
Diseases Genetic Diseases, X-Linked