MIAME COMPLIANCE CHECKLIST:
Gibson, Berger, Harshman, Kopp, Vacha, Nuzhdin, Wayne (2003)
Extensive Sex-Specific Non-Additivity of Gene Expression in Drosophila melanogaster
Experiment Design
Type of Experiment:
Quantitative analysis of inheritance of gene expression levels
in Drosophila melanogaster
Experimental Factors:
Genotype:
Sex:
2 isogenic parents (Oregon R; Russian 2b)
2 reciprocal F1s (O2b and 2bO)
Male versus Female
Number of Hybridizations:
28
Type of Reference:
None
Hybridization Design:
Complete loop with 7 replicates: see
http://statgen.ncsu.edu/ggibson/SupplInfo/NG03_SFig1.pdf
Quality Control:
Each of the 8 Samples (4 genotypes by 2 sexes) consists of several
hundred flies, so no individual variance measured
Each Sample labeled in 3 separate reactions per dye (8x3x2 = 48
total), and these were pooled before hybridization
to reduce labeling effects
Each comparison of sample types balanced for the two dyes
URL for Supplementary Data:
http://statgen.ncsu.edu/ggibson/SupplInfo/SupplInfo3.htm
Samples Used, Extract Preparation, and Labeling
Biological Samples:
Drosophila melanogaster (fruitfly)
Oregon R and 2b Stocks from Dr Sergey Nuzhdin in Fall 2002
Nearly isogenic parental lines derived by repeated backcrossing
F1s generated by crossing of multiple parents
(Oregon female by 2b male yields O2b; and vice verse for 2bO)
All fly rearing in Laboratory of Greg Gibson at NCSU
Biological Manipulations:
Whole adult flies, 7-10 days old, kept on 12 hour light/dark cycle
Sexes separated 4-7 days after eclosion (hence mostly mated)
Standard cornmeal plust yeast bottles for rearing
Flies snap frozen in mid-afternoon to control for diurnal rhythm
Hybridization Protocol:
Standard Agilent Protocol with Custom Agilent Chip
All hybridizations performed at Agilent facility in Palo Alto
February 14 and 15, 2003
Labeling Protocol:
Also Standard Protocol with Agilent Kit #G2554A
External controls:
None
Measurement Data and Specifications
Type of Data:
Raw background-subtracted fluorescence intensity transformed to
log base 2 scale, and dye effect normalized by loess transformation
(Columns BZ and CA of attached data files)
Scanning:
Agilent Scanner #G2565BA and associated software
Data files:
28 raw data files (Agilent) in tab-delimited text format
1 text file suitable for import into SAS or other statistical
software lists every log2 measurement before and after
ANOVA Step 1 below, with (28x2x21929 rows)
1 TIFF showing a typical image
Data transformation:
2-step mixed model ANOVA:
- Step 1: remove hybridization and dye effects
- Step 2: evaluate gene-specific effects of genotype, sex, and
genotype-by-sex interaction
Final Gene Expression Table:
http://statgen.ncsu.edu/ggibson/SupplInfo/NG03_STable.xls
Worksheet 3: All probes
Worksheets 1 and 2: Processed significant data for both sexes
Array Design
General:
60 mer oligonucleotides synthesized in situ on glass slides by
Agilent Technologies using phosphoramidite chemistry
Drosophila Design Custom designed for UC Davis, but
now commercially available
Features:
Exact feature sequences s and gene identifiers indicated in
columns F and J of each of the raw data files