Similar protocols

Protocol publication

[…] About 50 flies (w1118;+;Orco-Gal4/+) of both sexes were kept in each vial for 3 days. Female flies were then transferred to a new food vial (control fed flies) or a vial with a Kimwipe saturated by water (starved flies). 12 hr later, antennae were collected from these female flies. Dissection was performed in the morning at the same time to minimize circadian difference. Antennae from 200 flies were collected for each condition and total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA). Libraries were prepared using Illumina's mRNA sequencing kit and further purified using AMPure XP beads (Agencourt). Sequencing was performed at UCSD's BIOGEM facility on an Illumina GA2 sequencer. For each of the biological conditions, over 80 million 36 bp reads were generated from two lanes. Reads were aligned to the Drosophila genome (dm3 assembly) using TopHat (), allowing up to three mismatches with the reference sequence. Transcripts were then assembled against FlyBase (release 5.39) gene annotations and their abundances were calculated using Cufflinks (). In total, over 136 million reads were mapped to protein-coding genes. For differential expression analysis, raw gene counts were generated using HTSeq () software and then normalized for the difference in sequencing depth between the two conditions. Probability values were calculated on raw counts using the Fisher's exact test as computed by the edgeR package () (R software environment). […]

Pipeline specifications

Software tools TopHat, Cufflinks, HTSeq, edgeR
Databases FlyBase
Application RNA-seq analysis
Chemicals Tachykinins