gender: female strain: Df(2L)ED475/+ tissue: whole flies developmental stage: 5 day old mated adults
Treatment protocol
We collected replicates for experiments by outcrossing the balancer chromosome with the original wild-type stock (w1118) as follows; 18-24 vials were setup with 15 less than 8 day old virgin w1118 females paired with 5 less than 8 day old males of a particular deficiency line per vial. Df/+ female and males were placed together in vials at less than 60 per vial and then aged for five days. On day five, flies were anesthetized with CO2, sorted into males and females, then placed into 1.5 ml Eppendorf tubes and flash frozen on dry ice. All flies aged together for 5 days constituted a replicate. Flash frozen flies were stored in -80°C before RNA extraction.
Growth protocol
Flies were grown under constant light, temperature, and humidity (25°C; 60% relative humidity) on San Diego Stock Center cornmeal media (DSSC, 2008)
Extracted molecule
total RNA
Extraction protocol
Flies were immersed in 250 μl of TRIzol® (Invitrogen, Carlsbad, California) and thoroughly homogenized using a 1.5 ml RNAase free pestle (Kimble-Chase, Vineland, New Jersey) fitted to a Pellet Pestle Motor (Sigma-Aldrich, St. Louis, Missouri). After initial homogenization, 550 μl of TRIzol® was added and the solution was homogenized by hand until completely homogeneous. Chloroform (160 μl) was added to the homogenate and then the sample was centrifuged at 12,000 g for 15 minutes at 4°C. The aqueous layer was removed, precipitated with isopropyl alcohol and glycogen (8 μg), then centrifuged for 10 minutes at 4°C to pellet the RNA. The supernatant was removed and the RNA pellet was washed with fresh 75% ethanol. The pellet was dried briefly and then dissolved in 50-100 μl DEPC treated H2O. mRNA was enriched from total RNA by poly A+ selection using a QIAGEN Oligotex mRNA kit (Valencia, California) following manufacture instructions. mRNA was eluted from small spin columns twice with hot 30 μl buffer OEB. mRNA quality and quantity was determined by Bioanalyzer (Agilent, Santa Clara, California) and 260/280 ratio from Nanodrop ND-1000 UV spectrophotometer.
Label
Cy5
Label protocol
Microarrays were used in a two-color format with mRNA labeled with Cy3 or Cy5. Three or four biological replicates for each sex were labeled and hybridized as follows. For each sample of mRNA, 200 ng was simultaneously reverse-transcribed and labeled with dye along with three Arabidopsis in vitro transcribed spike-in controls (AY11387 at 1:1 ratio between Cy3 and Cy5 at 1 ng/ul; AF33972 at 1:2 ratio between Cy3 and Cy5 at 1:10 dilution (0.1ng/ul); and AY093727 at 1:1 ratio between Cy3 and Cy5 and 1:50 dilution (0.02ng/ul)). Reverse-transcription/labeling reactions consisted of 5 μl 5X First Strand Buffer (Invitrogen, Carlsbad, California), 1 μl 0.1 mM DTT (Invitrogen, Carlsbad, California), 3 μl of dye-mix containing Cy3 or Cy5 labeled nanomer primer (Trilink Biotechnologies, San Diego, CA), 1 μl RNase inhibitor (Ambion, Austin, Texas), 5 μl H2O plus Arabidopsis spike-in controls, and 1 μl Super-script II reverse transcriptase (Invitrogen, Carlsbad, California). Labeling reactions were incubated at 37°C for 2 hours then stopped by heat termination at 85°C for 5 min. Labeling reactions were purified using Clonetech columns (TE-10), Clonetech, Mountain View, California) following manufacture instructions. Samples to be co-hybridized onto one microarray were pooled and precipitated with 90 μl DEPC treated H2O, 1 μl glycogen (1 mg/ml), 60 μl 5M ammonium acetate, and 300 μl cold 100% EtOH and by cold centrifugation at max speed for 20 min. Different sexes were never co-hybridized and we tried to have all replicates for a particular Df/+ line be hybridized on the same glass slide. Probe pellets were re- suspended in 6.6 μl Sample Tracking Controls (Roche Nimblegen Inc., Madison, Wisconsin), 4.8 μl H2O, 12 μl warm 2X Hyb Buffer (250 μl formamide, 250 μl 20X SSC, 20 μl 10% SDS, 5 μl tRNA (2mg/ml)), 0.3 μl Nimblegen feducial Cy3 CPK6 and 0.3 μl Cy5 CPK6. Hybridization cocktails were heated to 95°C for 5 minutes then placed at 42°C until loaded onto arrays for hybridization.
Hybridization protocol
Microarray glass slides were fitted with 12X mixers according to standard Nimblegen protocols, warmed to 42°C on a MAUI hybridization station (BioMicro Systems, Salt Lake City, Utah), and 7 μl of hybridization cocktail was loaded onto each microarray through the fill port. Vent and fill ports were sealed and arrays were loaded onto the MAUI station. Mix mode B on the MAUI hybridization station was started and samples were hybridized for 16 hours at 42°C in an ozone free chamber. The ozone free chamber consisted of a homemade Lexon box (182 cm x 67 cm x 67 cm). Two NoZone® Ozone Scrubbers (SciGene Corporation, Sunnyvale, CA) were used to pump ozone free air into the box. Ozone levels were measured using a Aeroqual Series 500 ozone monitor (Aeroqual Limited, Auckland, New Zealand) and found to be lower compared to levels of ozone external to the box. Hybridization, washing, and scanning were all performed in the ozone free chamber. Microarray glass slides were washed using the following modified Nimblegen protocol. A glass slide with mixer was submerged for 10 seconds in warm (42°C) Buffer I (1 L H2O, 10 ml 20X SSC, 20 ml 10 % SDS, 100 μl 1 M DTT) and the mixer was removed. Slides were placed in a fresh beaker of 42°C Buffer I and agitated for 2 minutes, submerged for 1 minute in room temperature Buffer II (1000 L H2O, 10 ml 20X SSC, 100 μl 1 M DTT), followed by submerging for 15 sec in Buffer III (1 L H2O, 2.5 ml 20X SSC, 100 μl 1 M DTT). Microarray slides were placed in a ChipMate Mini Slide Centrifuge (Tomy Tech USA Inc., Menlo Park, California) and spun for 1 minute to dry. Dried microarray slides were covered with aluminum foil until scanning.
Scan protocol
Slides were scanned on a Axon GenePix® 4200 AL (Molecular Devices, Sunnyvale, California) scanner controlled by GenePix® 6.0 software. Laser PMT settings were calibrated using a dilution series of Cy3 and Cy5 dyes. For microarray data collection, Cy3 and Cy5 channels were balanced by visual inspection of histograms for Cy3 and Cy5 channels and if necessary by manually changing PMT settings for each laser. Each scan was conducted at 5 μm resolution with no line averaging and saved as a TIFF file.
Description
F_ED475_R1
Data processing
TIFF flies were imported into Nimblescan v. 2.4.27 (Roche Nimblegen Inc., Madison, Wisconsin) for processing. We used the burst multiplex image function to separate each channel of the 2-color 12-plex image into 24 array single color images. Probe intensities for each subarray were extracted for probes mapping to Flybase Drosophila melanogaster r. 5.5 FBgn numbers using the autogrid function and then feature intensities were exported as individual pair files in Nimblescan. We updated the annotation to reflect Flybase D. melanogaster r. 5.29 but broad conclusions derived from our analysis were not dependent on differences in annotation. Cy3 channel images were then analyzed for array to array contamination using sample tracking analysis in Nimblescan. Sample tracking provides a report of the sample tracking controls that were included with each array hybridization. All sample tracking analyses were co-linear with sample tracking controls indicating no contamination occurred across microarrays within a glass slide. Raw intensities in pair files for each probe were log2 transformed. Pearson’s r and Spearman’s rho correlation coefficients were calculated for all pairwise comparisons within replicates in order to identify outlier hybridizations. Replicates that had Spearman’s rho greater than 0.80 were retained for subsequent normalization and analysis. This eliminated 6 channels of data giving 134 channels for subsequent analysis.
Normalization was performed by loading the remaining single array pair files into Nimblescan v. 2.4.27 (Roche Nimblegen Inc., Madison, Wisconsin) and then normalizing all data together using Robust Multichip Averaging (RMA; Irizarry et al. 2003) both with and without background correction. Results with background correction are depoisted here.
