Similar protocols

Pipeline publication

[…] ed out with an Applied Biosystems 3730 Thermoycler and SYBR Green, or with a BioRad C1000 Touch Thermal Cycler and SsoAdvanced SYBR Green according to manufacturers' instructions., 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., We thank Judy Kassis, Yuzhong Cheng, Susan Younger, Bill Sullivan, Jim Jaynes, Michael Levine, David Casso, Sougata Roy, and Markus Noll for generously helping with reagents and advice, and the Bloomington Drosophila Stock Center for fly lines.] […]

Pipeline specifications

Software tools TopHat, Cufflinks, SAMtools, DAVID