After 24 h incubation with N and Fe substrates, 2 L of seawater from each incubation were collected before sunrise and filtered onto 0.2 µm Supor membrane filters (Pall Corp., Ann Arbor, Michigan, U.S.A.) using peristaltic pumps. Filters were flash frozen in liquid nitrogen immediately after filtering and stored at -80°C until processing in the lab.
Growth protocol
Environmental samples were collected during the Nitrogen Effects on Marine microOrganisms (NEMO) Cruise NH1417 in 2014. Seawater collected from 25 m depth at Stn. 38 on 24 August was used in on-deck perturbation experiments with different nitrogen substrates (nitrate, ammonium, urea, iron, nitrate with iron, and also with filtered deep water collected from 600 m depth). Experimental bottles were incubated on deck for 48 h, with RNA collected at 24 h for all treatments and also at the start of the incubation for controls (no substrate added). Experimental setup and physiological responses to added nutrients observed at 48 h are described in detail in Shilova et al. 2017.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from environmental samples using DirectZol (Zymo Research). DNA was removed in a solution using RNase-Free DNase Kit (Qiagen), and RNA was purified again with RNA Clean & Concentrator™-25 (Zymo Research) according to the manufacturer’s protocol. The RNA quality and quantity were 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 as described in Shilova et al., 2014.
Label
Cy3
Label protocol
Provisional: 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 transferred 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
Provisional: 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 G2600D (Serial #: SG13134301) and using the Agilent scanning protocol GE1_1200_Jun14 (Feature Extractor software version 11.5.1.1).
Description
environmental sample
Data processing
All microarray analyses were done using the MicroTOOLs R package (ver. 1.1; available at https://www.jzehrlab.com). The transcription values for each gene were obtained by robust multi-array averaging 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 and gene sets (described in our publication), but those steps do not affect the values in the sample matrices.
