|
| Status |
Public on Sep 03, 2018 |
| Title |
ESP_Sep16_1700_id_3053 |
| Sample type |
RNA |
| |
|
| Source name |
North Pacific Subtropical Gyre (NPSG) surface water (24 m)
|
| Organism |
uncultured marine microorganism |
| Characteristics |
category: in situ
water_type: ESP
date: 16-Sep
|
| Treatment protocol |
Environmental samples were filtered using 0.2 micron Sterivex cartridges. Filters were immediately flash frozen with liquid nitrogen during the cruise. After the cruise, filters were shipped to UC Santa Cruz where they were stored at -80 Celsius.
|
| Growth protocol |
Environmental samples were collected during the BioLINCS Cruise from 6-21 September 2011. In situ samples were collected with an Environmental Sample Processor (ESP). Mixing experiment samples were collected from CTD casts.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RiboPure RNA purification kit with the addition of a bead-beating step during TRI Reagent extraction as described in Shilova et al., 2014. DNA was digested using the RNase-Free DNase Kit (Qiagen) according to the manufacturer’s protocol and RNA quality and quantity was evaluated using the Agilent BioAnalyzer RNA Nano Kit and Qiagen Qubit. All samples with an RNA Integrity Number greater than 9 were processed for microarray analyses.
|
| Label |
Cy3
|
| Label protocol |
cDNA was labeled at the Roy J. Carver Center for Genomics (The University of Iowa, USA) using the Agilent SureTag DNA Labeling Kit (Cat# 5190-3400) and by following a protocol based on Agilent Oligonucleotide Array‐Based CGH for Genomic DNA Analysis: Enzymatic Labeling for Blood, Cells, or Tissues [Version 7.3 March 2014]. For each sample the following steps were performed. 0.5 ug of cDNA was diluted to 19.2 ng/ul [26.0 ul total]. The diluted cDNA was then denatured and annealed with random primers: cDNA was combined with 5.0 ul of random primers and the resulting 31.0 ul sample was incubated at 95 degrees Celsius for 5 min, then immediately placed on ice for 5 min, and then centrifuged at 6kxg for 1 min. 19.0 ug of labeled master mix was prepared (5x reaction [10.0 ul], 10x dNTP [5.0 ul]; cyanine [3.0 ul]; Exo-Klenow [1.0 ul]) and then pipetted into the sample. The sample was briefly centrifuged, then incubated at 37 degrees Celsius for 2 hrs and at 65 degrees Celsius for 10 minutes, and then transfered on ice (and possibly stored overnight at -20 Celsius). Labeled cDNA was then cleaned using the Agilent SureTag DNA Labeling Kit, which included purification columns. A Nanodrop spectrophotometer was used to check that the yield and specific activity of Cy3 were within expected ranges.
|
| |
|
| Hybridization protocol |
Cy3-labeled cDNA was hybridized at the Roy J. Carver Center for Genomics (The University of Iowa, USA) using a Gene Expression Hybridization Kit (Cat# 5188‐5242) and following a protocol based on One-Color Microarray-Based Gene Expression Analysis: Low Input Quick Amp Labeling [Version 6.7, September 2014]. From each Cy3-labeled sample, a volume equivalent to 3.0 ug of target DNA was taken and added to water to a total volume of 44.00 ul. To each sample was added 66.0 ul of hybridization master mix (10x GE blocking agent [11.0 ul], 2x Hi-RPM Hyb Buffer [55.0 ul]) for a total volume of 110 ul. The sample was mixed, then incubated at 95 degrees Celsius for 3 min, then placed on ice, and then briefly centrifuged. From each sample 100 ul was taken and loaded onto a MicroTOOLs microarray (4 arrays per slide). Arrays were hybridized at 65 degrees Celsius and for ~18 hours. After hybridization, arrays were washed using the Gene Expression Wash Buffer (Cat# 5188‐5327).
|
| Scan protocol |
Microarrays were scanned at the Roy J. Carver Center for Genomics at the University of Iowa using an Agilent SureScan Microarray Scanner G2600DA (UI Tag: 607105, Serial #: SG13134301) and using the Agilent scanning protocol GE1_1105_Oct12 (Feature Extractor software version 11.5.1.1).
|
| Description |
environmental sample
|
| Data processing |
All microarray analyses were done using the MicroTOOLs R package (ver. 1.0; available at https://www.jzehrlab.com). The transcription values for each gene were obtained by robust multi-array average of hybridization values for all probes and quantile normalization across all samples. Additional data processing steps were performed for gene detection and determination of differentially expressed genes (described in our publication), but those steps do not affect the values in the sample matrices.
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| |
|
| Submission date |
Jan 16, 2018 |
| Last update date |
Sep 03, 2018 |
| Contact name |
Jonathan David Magasin |
| E-mail(s) |
jmagasin@ucsc.edu
|
| Organization name |
UC Santa Cruz
|
| Department |
Ocean Sciences
|
| Lab |
Dr. Jonathan Zehr Lab
|
| Street address |
1156 High Street
|
| City |
Santa Cruz |
| State/province |
CA |
| ZIP/Postal code |
95062 |
| Country |
USA |
| |
|
| Platform ID |
GPL24371 |
| Series (1) |
| GSE109218 |
Effects of nutrient enrichment on surface microbial community gene expression in the oligotrophic North Pacific Subtropical Gyre |
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