Dataset: Microarray gene transcription
Deployment: NH1417

Methodology:

Nutrient amendments incubation experiments were conducted during the Nitrogen Effects on Marine microOrganisms (NEMO) Cruise NH1417 in 2014 and described in Shilova et al. (2017). Briefly, 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). Control bottles with no added nutrients were included. The experiment setup was done under trace metal clean conditions. 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.

Sampling and analytical procedures:

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 -80C until processing in the lab. 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 manufacturers’ protocols. 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. 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]. Microarray platform GPL24371 Agilent-073391 MicroTOOLs_171K_oligo_v2.0 was used in this study. 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).