trancriptome profiling of pennellii ILs

Hybridization: 10-2-2/M82

Hybridization protocol
Hybridization solutionGenisphere Hybridization Solution
Solution concentration0.25m NaPO4, 4.5%SDS, 1mm EDTA, 1x SSC, 2x Denhardt's Solution
Blocking agentssDNA and Oligo dT
Wash procedure3x15 minutes:
1) 2x SSC + 0.2% SDS at 60 C.
2) 2x SSC at 22 C.
3) 0.2x SSC at 22 C
Labeled Target Quantity3 ul cDNA
Hybridization Time12-16 hours
Hybridization Volume60 ul
Hybridization Temperature60C
Hybridization Instrument50 ml tube in 60C chamber

Scan information
Scan hardwareScan Array 5000
scan softwareScan Array ver 3.1

Image analysis information
SoftwareImaGene ver 5.5.4
Analysis algorithm1) Images
Cy5 = Red Cy3 = Green

2) Grid Flexibility
Enforce grid constraints = yes
Local flexibility = 6.0 pixels
Flexibility = 75%

3) Quality Control
Empty spots = no
Poor spots = yes
Find negative spots = no

4) Measurements
Background buffer = 2.25
Background width = 5.0
Auto seqmentation = no
Signal low cutoff = 20%
Signal high cutoff = 100%
Background low cutoff = 0%
Background high cutoff = 88%

5) Poor Spot Parameters
Contamination of Background/Signal = no
Ignored pixels % = yes (setting = 69)
Open perimeter = no
Shape regularity = no
Area to perimeter = yes (setting = 0.6)
Off-set value = yes (setting = 17.5)

Normalization information
Normalization softwaremarray package under R
Normalization algorithmPrint-tip specific Lowess Normalization
Normalization strategyTotal Array