|
| Status |
Public on Jun 20, 2013 |
| Title |
Water column DNA_ETSP_Station 12_20m |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Universal Internal Standard + 70-mer
|
| Organism |
synthetic construct |
| Characteristics |
sample type: reference
|
| Extracted molecule |
other |
| Extraction protocol |
DNA was extracted from the filters using the PureGene DNA kit (Gentra, Minneapolis, MN) or the AllPrep DNA/RNA Mini Kit (Qiagen Sciences, Maryland, USA) with slight modifications (as in Ward 2008).
|
| Label |
Cy5
|
| Label protocol |
Hybridization targets were prepared by digesting environmental DNA with a four cutter restriction enzyme. The digested environmental DNA were labeled with amino-allyl-dUTP (Ambion) during linear amplification using random octamers and a Klenow polymerase (Applied Biosystems). The reaction contained 3.96 mM d(AGC)TP, 0.44 mM dTTP, and 4.84 mM dUaa and was amplified for 3 h at 37°C. The Klenow product was purified and conjugated with Cy3. The Cy3-labelled target (1000 ng) was combined with 2x hybridization buffer (1x final concentration; Agilent) and 0.25 pmol of a Cy5-labelled complementary 20-mer standard oligonucleotide and incubated at 95°C for 5 min before being cooled to room temperature.
|
| |
|
| Channel 2 |
| Source name |
Total DNA from water column, ETSP station 12, 20m
|
| Organism |
marine metagenome |
| Characteristics |
location: Eastern Tropical South Pacific (ETSP) Station 12
depth: 20 m
sample type: environmental
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from the filters using the PureGene DNA kit (Gentra, Minneapolis, MN) or the AllPrep DNA/RNA Mini Kit (Qiagen Sciences, Maryland, USA) with slight modifications (as in Ward 2008).
|
| Label |
Cy3
|
| Label protocol |
Hybridization targets were prepared by digesting environmental DNA with a four cutter restriction enzyme. The digested environmental DNA were labeled with amino-allyl-dUTP (Ambion) during linear amplification using random octamers and a Klenow polymerase (Applied Biosystems). The reaction contained 3.96 mM d(AGC)TP, 0.44 mM dTTP, and 4.84 mM dUaa and was amplified for 3 h at 37°C. The Klenow product was purified and conjugated with Cy3. The Cy3-labelled target (1000 ng) was combined with 2x hybridization buffer (1x final concentration; Agilent) and 0.25 pmol of a Cy5-labelled complementary 20-mer standard oligonucleotide and incubated at 95°C for 5 min before being cooled to room temperature.
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized to duplicate arrays by overnight incubation at 64°C and washed.
|
| Scan protocol |
The arrays were scanned with a laser scanner (Molecular Devices 4200).
Images were analyzed with GenePix Pro 6.0 software (Molecular Devices).
|
| Description |
E12.20m
This Sample represents 2 replicates.
DNA extracted from sterivex filter used to create 2 targets to run on duplicate arrays.
|
| Data processing |
Quality control was employed to remove noise by eliminating features 1) in which the Cy3 signal was not at least twice the value of the control probes, and 2) in which the Cy3/Cy5 could not pass a Z test to find out the outlier values. The Z test was performed by calculating Zi = ri/(s/square root of 3), i = 1, 2, or 3, where ri represents Cy3/Cy5, s the standard deviation of the triplicate Cy3/Cy5 values. A feature is considered as an outlier if the Z is greater than 1.9 (CI = 80%).
Cy3/Cy5 fluorescence intensities were standardized to the highest Cy3/Cy5 fluorescence across the AOA probe set (normalized fluorescence ratio; FRN).
|
| |
|
| Submission date |
May 12, 2013 |
| Last update date |
Jun 20, 2013 |
| Contact name |
Xuefeng Peng |
| Organization name |
Princeton University
|
| Department |
Geosciences
|
| Street address |
Guyot Hall
|
| City |
Princeton |
| State/province |
NJ |
| ZIP/Postal code |
08544 |
| Country |
USA |
| |
|
| Platform ID |
GPL17151 |
| Series (1) |
| GSE46851 |
Community composition of ammonia-oxidizing archaea from surface and anoxic depths of oceanic oxygen minimum zones |
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