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Sample GSM2935514 Query DataSets for GSM2935514
Status Public on Sep 03, 2018
Title ESP_Sep16_500_id_3052
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.
 
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

Data table header descriptions
ID_REF
VALUE Normalized log2 transcript levels for genes

Data table
ID_REF VALUE
G021500900 4.668992968
G021500901 2.597744267
G021500902 3.264569631
G021500903 5.69131265
G021500904 8.859166921
G021500905 10.3627057
G021500906 6.377815279
G021500907 5.493340715
G021500908 1.419077416
G021500909 3.005319274
G021500910 3.325822856
G021500911 3.860127096
G021500913 4.798190126
G021500914 4.883984947
G021500915 2.951593738
G021500916 4.754294025
G021500917 5.935648474
G021500918 6.019688402
G021500919 5.652265115
G021500920 5.65467829

Total number of rows: 19525

Table truncated, full table size 464 Kbytes.




Supplementary file Size Download File type/resource
GSM2935514_SG13134301_257339110007_S001_GE1_1105_Oct12_1_3.txt.gz 8.4 Mb (ftp)(http) TXT
Processed data included within Sample table

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