|
| Status |
Public on Sep 12, 2013 |
| Title |
water column DNA_Arabian Sea_Station 1_60m |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Universal Internal Standard + 70-mer
|
| Organism |
synthetic construct |
| Characteristics |
sample type: Universal Internal Standard + 70-mer
|
| 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 colum
|
| Organism |
marine metagenome |
| Characteristics |
location: Arabian Sea Station 1
depth: 60 m
|
| 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 Gene Pix Pro 6.0 software (Molecular Devices)
|
| Description |
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%).
|
| |
|
| Submission date |
Sep 11, 2013 |
| Last update date |
Sep 12, 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 |
GPL17721 |
| Series (1) |
| GSE50787 |
Community composition of bacteria involved in fixed nitrogen loss in the water column of two major oxygen minimum zones in the ocean |
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