genotype: XY nucleic acid: RNA autosome: c167.4/ste x origin: c167.4 Sex: M rep: 3
Extracted molecule
total RNA
Extraction protocol
Flies were freeze dried at -20° overnight prior to homogenization. Dried flies were ground to a fine powder using a GenoGrinder (Maximum, three minutes, repeated twice). Trizol (1 ml) was added to each homogenized sample and mixed thoroughly in the GenoGrinder (Max, 3 minutes). Samples were transferred to a new tube, 1 ul linear acrylamide was added to each and samples were then incubated at room temperature for 5 minutes. RNA was extracted using a standard Trizol extraction protocol: phase separation using 0.1 vol BCP, RNA precipitation with isopropanol, 70% ethanol wash and resuspension in 80 ul DEPC H2O. Concentration was measured using a NanoDrop and up to 30 ug RNA per sample was treated with DNase I in 100 ul reaction volumes for 30 minutes at 37° (reaction mix: 4U Cloned DNase I TaKaRa 2220A, 80U Promega Recombinant RNasin N2515, in 1X TaKaRa Cloned DNase I Buffer II). Samples were cleaned prior to concentration using the Qiagen RNeasy Mini Kit (Cat. # 74104) following the manufacturer’s standard protocol with 30 ul DEPC H2O elutions (run through the column twice). RNA quality was examined using BioAnalyzer RNA 6000 Nano chips and all samples were found to be of good quality. Genomic DNA was isolated from 35-40 flash frozen females using the AllPrep Mini Kit (Qiagen) following standard manufacturer’s protocols. Samples were concentrated by standard ethanol precipitation and resuspended in 31 ul DEPC H2O.
The prepared target samples were then hybridized using the Hybridization, Wash and Stain Kit (P/N 900720) following the manufacturer’s protocol for the Fluidics Station 450 with protocol FS450_0001.
Scan protocol
Arrays were scanned using an Affymetrix® 7G scanner.
Description
4899_R3_F_RNA.CEL
Data processing
Signals were extracted from the scans using the apt-cel-extract program of the Affymetrix Powel Tools® (version 1.10.2) suite. GC bin control probes provide an estimate of non-specific hybridization (Affymetrix 2005) and help to assess the overall quality of the hybridization. A GC bin control is a standard Affymetrix® control based upon the number of G/C bases (from 3 to 24) in the 25 mer probe. None of the GC bin control probes align to the D. melanogaster or D. simulans reference genomes. Individual probes were classified according to their GC content and matched to the corresponding GC bin controls. A probe was considered detected when signal strength was higher than the median intensity of the corresponding GC band controls. Detection above background (DABG) was calculated at the individual probe level. To correct for the background noise and to normalize the probe signals, each probe was classified into a GC bin and the 5 percentile signal for that GC bin was subtracted from the probe. The expression of a probe set was estimated by the natural log of the arithmetic mean of individual probe signals.
