Stage 15-16 Drosophila melanogaster embryos were collected on apple juice/agar plates at 25C.
Extracted molecule
total RNA
Extraction protocol
Embryos were dechorionated for 2 min in 50% bleach and rinsed well with embryo wash buffer (0.04% Triton X-100; 0.7% NaCl) and distilled water. Embryos were transferred to a 7 mL Dounce Homogenizer on ice containing 5 mL ice-cold Schneider media supplemented with 3 mM EGTA and homogenized gently with the loose pestle for 8 – 10 strokes. Debris was pelleted in a 15 mL conical tube for 3 min at 50 x g and the supernatant decanted to a fresh conical tube. Cells were pelleted at 350 x g for 5 min, resuspended in 1.5 mL cold PBS (pH 7.4) and passed through a 70 micron filter. All centrifugation steps were performed at 4ºC. Cells were stained with 7-Amino-actinomycin D (7-AAD) prior to Fluorescence Activated Cell Sorting (FACS) to identify membrane-intact live cells. Dissociated cells were sorted on a MoFlo high speed sorter (Beckman Coulter) for FACS of GFP positive cells. Gating was performed by first excluding sub-cellular debris from a plot of light scatter characteristics. Further gating on pulse width was performed to exclude undesired large particles, clusters of cells and small debris. Finally, GFP was plotted against yellow autofluorescence and single GFP positive cells were isolated for further analysis. GFP and autofluorescence were both excited with 488 nm laser light at 200 mW from a water-cooled argon ion laser (Coherent). GFP fluorescence was collected behind a 530/40 bandpass filter and autofluorescence behind a 580/30 filter on the FL1 and FL2 detectors, respectively. Approximately 150,000 to 300,000 cells were sorted for each biological sample (timed collections), with cell dissociation taking ~15 min and FACS occurring immediately following dissociation for ~0.5 – 1h. Cells were sorted directly into TriZol, and RNA was isolated and then amplified using MessageAmp II aRNA amplification kit (Ambion).
Label
Cy3
Label protocol
Concentration and quality of RNA were determined on the Bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA). For array analysis, labeled aRNA was prepared starting with ≤ 1 µg of total RNA using the Amino Allyl MessageAmp II aRNA Amplification Kit (Applied Biosystems/Ambion, Austin, TX) according to the manufacturer’s specifications. 5 µg amino allyl-modified aRNA was coupled with Cy dyes (CyDye Post-Labeling Reactive Dye Pack, GE Heathcare, RPN 5661). Labeling reactions were quenched with hydroxylamine, and uncoupled dye material removed with aRNA Filter Cartridges supplied in the MessageAmp II kit. Labeled aRNA was fragmented to 60-200 nts using Ambion RNA Fragmentation Reagents (P/N AM8740) according to manufacturer’s instructions.
Stage 15-16 Drosophila melanogaster embryos were collected on apple juice/agar plates at 25C.
Extracted molecule
total RNA
Extraction protocol
Embryos were dechorionated for 2 min in 50% bleach and rinsed well with embryo wash buffer (0.04% Triton X-100; 0.7% NaCl) and distilled water. Embryos were transferred to a 7 mL Dounce Homogenizer on ice containing 5 mL ice-cold Schneider media supplemented with 3 mM EGTA and homogenized gently with the loose pestle for 8 – 10 strokes. Debris was pelleted in a 15 mL conical tube for 3 min at 50 x g and the supernatant decanted to a fresh conical tube. Cells were pelleted at 350 x g for 5 min, resuspended in 1.5 mL cold PBS (pH 7.4) and passed through a 70 micron filter. All centrifugation steps were performed at 4ºC. Cells were stained with 7-Amino-actinomycin D (7-AAD) prior to Fluorescence Activated Cell Sorting (FACS) to identify membrane-intact live cells. Dissociated cells were sorted on a MoFlo high speed sorter (Beckman Coulter) for FACS of GFP positive cells. Gating was performed by first excluding sub-cellular debris from a plot of light scatter characteristics. Further gating on pulse width was performed to exclude undesired large particles, clusters of cells and small debris. Finally, GFP was plotted against yellow autofluorescence and single GFP positive cells were isolated for further analysis. GFP and autofluorescence were both excited with 488 nm laser light at 200 mW from a water-cooled argon ion laser (Coherent). GFP fluorescence was collected behind a 530/40 bandpass filter and autofluorescence behind a 580/30 filter on the FL1 and FL2 detectors, respectively. Approximately 150,000 to 300,000 cells were sorted for each biological sample (timed collections), with cell dissociation taking ~15 min and FACS occurring immediately following dissociation for ~0.5 – 1h. Cells were sorted directly into TriZol, and RNA was isolated and then amplified using MessageAmp II aRNA amplification kit (Ambion).
Label
Cy5
Label protocol
Concentration and quality of RNA were determined on the Bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA). For array analysis, labeled aRNA was prepared starting with ≤ 1 µg of total RNA using the Amino Allyl MessageAmp II aRNA Amplification Kit (Applied Biosystems/Ambion, Austin, TX) according to the manufacturer’s specifications. 5 µg amino allyl-modified aRNA was coupled with Cy dyes (CyDye Post-Labeling Reactive Dye Pack, GE Heathcare, RPN 5661). Labeling reactions were quenched with hydroxylamine, and uncoupled dye material removed with aRNA Filter Cartridges supplied in the MessageAmp II kit. Labeled aRNA was fragmented to 60-200 nts using Ambion RNA Fragmentation Reagents (P/N AM8740) according to manufacturer’s instructions.
Hybridization protocol
Slides were hybridized with a mixture of Cy3 and Cy5 labeled aRNA probes. Hybridizations were performed at 63°C overnight under standard conditions (3X SSC, 0.2 mg/ml salmon sperm DNA, 25 mM HEPES, 0.25% SDS) and slides were washed at room temperature successively with 0.6X SSC/0.03% SDS and then 0.06X SSC prior to scanning.
Scan protocol
Microarray images were acquired with a GenePix 4000B scanner (Axon Instruments, Foster City, CA). For image analysis GenePix pro 6.0 software was used (Axon Instruments).
Data processing
Data files were uploaded to Acuity version 4.0 (Molecular Devices), where the raw data were log-transformed, normalized using Lowess normalization, filtered for quality spots (RgnR2[635/532]>0.5; F532median - B532 >= 150; F635median - B635 >= 150; problematic or flagged spots removed). Mean values from dye-swap experiments were used for further analysis. Oligonucleotide probes were mapped to genes and transcripts defined in Flybase (version FB2009_07, r5.20, released August 10, 2009) (http://flybase.org/) (Tweedie et al. 2009).
