Title: Affymetrix CEL analysis. Description:
nucleic acid labeling protocol
Labeling of samples was carried out according to the manufacturer’s standard protocol
normalization data transformation protocol
the .CEL files were analysed using the method RMA.
nucleic acid extraction protocol
RNA pools from the tracheae (including anterior spiracles) of 50 3rd instar larvae were isolated, purified with RNA clean-up purification kits (Macherey Nagel), and DNase treated. The samples were controlled for fat body contamination by RT-qPCR on Fat body protein P6 (Fbp2). RNA quality was controlled on Agilent 2100 Bioanalyzer chips. As the quality of some samples was not good enough after this first purification, RNA of all samples was ethanol precipitated to pass Bioanalyzer quality control. (Parameters: Extracted product = total_RNA, Amplification = RNA polymerases)
Larvae were infected by adding 500uL of concentrated bacterial suspension (OD600=0.2 after 1/1000 dilution) in the fly vial. Third instar larvae (recognized by the hand-shaped anterior spiracle) were dissected 24h later by gently pulling the posterior spiracles backwards until the whole tracheae went out. If needed, the anterior part of the tracheae was pulled out in a second similar step.
Drosophila were grown at 25degreesC in corn meal fly medium supplemented with live yeast. (Parameters: time unit = seconds, min temperature = 25, temperature unit = C, media = corn meal fly medium)