Computational protocol: An Essential Role for Zygotic Expression in the Pre Cellular Drosophila Embryo

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Protocol publication

[…] RNA was isolated from the progeny of two inbred, fully-sequenced lines from the Drosophila Genetic Research Panel : RAL-208 and RAL-555. Reciprocal crosses were made; RNA was from three pools, each pool from five embryos that were stages 3–6, 7 or 8. Every embryo was individually staged under a dissecting microscope. RNA was sequenced with the Illumina Genome Analyzer using protocols described in .Reads from each RNA-Seq sample were mapped to the reference D. melanogaster genome (FlyBase release 5.27; , ) using Bowtie and TopHat , and transcript abundances for annotated RNAs were called by Cufflinks . Data from each sample were normalized so that the total expression (reads per kb of sequence, per million mapped reads; RPKM) of autosomal genes was constant. We exported reads for every gene using SAMTOOLS and examined all reads for every gene to determine if they overlapped a polymorphism between RAL-208 and RAL-555 as described in DGRP release 1.0. If a read overlapped a SNP, we determined whether it was of maternal or paternal origin. The vast majority of SNP reads in genes could be assigned to maternal chromosomes. For highly expressed genes, between .1 and 1% of reads mapped to the paternal chromosome, an observation we attribute to sequencing errors. A small number of genes had roughly even numbers of maternal and paternal reads, which we attributed to zygotic transcription. […]

Pipeline specifications

Software tools Bowtie, TopHat, Cufflinks, SAMtools
Databases FlyBase
Organisms Drosophila melanogaster, Caenorhabditis elegans