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Sample GSM2367780

Query DataSets for GSM2367780

Status

Public on Feb 19, 2017

Title

Control_rep2

Sample type

SRA

Source name

RNA

Organism

Acaryochloris marina MBIC11017

Characteristics

treatment: control

Treatment protocol

Cultures under normal O2 levels were inoculated in 1-l Erlenmeyer glass flask containing 500 ml of medium, capped with a cotton stopper that permitted gas exchange. Thus, the O2 concentration of the gas phase inside the bottle was similar to atmospheric levels ~21% (v/v). Microoxic conditions were achieved by using a 500-ml two-necked round-bottom flask sealed tightly by a rubber stopper. Cultures were vacuumed, and refilled with pure nitrogen gas (99.95% purity) to ensure normal atmospheric pressure. We repeated this process several times, yielding a final O2 concentration of <0.2% inside the sealed culture flask. To maintain a microoxic condition, a positive pressure was created by bubbling nitrogen through the culture. A similar set-up was used for hyperoxic conditions, with the exception that pure O2 gas was used to refill the flask after vacuuming. Because the cells generate O2 under illumination, ongoing input of O2 gas to maintain the high-oxygen concentration was not required. However, to avoid pressure build-up, the flask was re-vacuumed and refilled with O2 gas every 48 h, as described above. The O2 concentration inside the flask remained within the range of 65–75% (v/v) during the experiment.

Growth protocol

Acaryochloris marina MBIC11017 was routinely kept in a culture room at 27°C under 15–30 µmol photons•m˗2•s˗1 of cool white light. Sterilized K+ES (artificial seawater), buffered with 25 mM TES at pH 8.0 was used as the culture medium for all three treatment groups. To make sure photosynthesis was not limited by CO2, NaHCO3 was dissolved in a small volume of autoclaved media, and injected into the enclosed culture flasks every two days (yielding an initial concentration of 0.375 mM). The initial cell density of all culture groups was adjusted to an optical density at 750 nm of 0.2. The cultures were shaken on an orbital flat-bed shaker at ~90 rpm.

Extracted molecule

total RNA

Extraction protocol

Acaryochloris cultures were harvested after five days from all three different treatments. The harvested cell pellets were mixed with TRIzol (TRIzol® Reagent, Life Technologies, Australia), and frozen immediately using liquid nitrogen. They were stored at ˗80°C for at least 60 min. The frozen samples were thawed in a water bath of 37°C, and spun down at 16,000×g for 5 min to eliminate cellular debris. This supernatant was mixed at a volume ratio of 4:1, with chloroform and spun down at 16,000×g for 10 min at 4°C. The upper layer, containing RNA and DNA, was carefully transferred to a new tube, without disturbing the white middle layer of solid components. The RNA was precipitated by addition of an equal volume of isopropanol and incubated at ˗20°C for at least 45 min. RNA pellets were washed with 70% (v/v) ethanol and the DNA removed using the Baseline-ZERO™ DNase kit (Epicentre, WI, USA), following the manufacturer's instructions. Prior to RNA quality assessment, the absence of DNA was confirmed by polymerase chain reaction (data not shown).
The quality of RNA was assessed on a 2100 Bioanalyzer (Agilent Technologies, CA, USA), using a RNA 6000 Nano RNA Kit (Agilent Technologies) to obtain an RNA integrity number of >8.0. Transcripts corresponding to rRNAs (5S, 16S and 23S) were reduced from the samples with a Ribo-Zero Kit (Epicentre), following the manufacturer's instructions. The remaining RNA was mixed with the fragmentation buffer, the RNA was fragmented into short fragments. Then cDNA was synthesized using random hexamers and the RNA fragments as templates. Short fragments were purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments were connected with adapters. After agarose gel electrophoresis, the suitable fragments were selected for PCR amplification as templates. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System were used in quantification and qualification of the sample library. At last, the library could be sequenced using Illumina HiSeqTM 2000. Further processing, including quality assessment, was undertaken prior to RNAseq analysis by Beijing Genomics Institution, China.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Control

Data processing

Raw data was processed using Rockhopper java based software with default parameters for paired-end sequencing files
Genome_build: Acaryochloris marina MBIC11017
Supplementary_files_format_and_content: Expression_data.xlsx (Differential expression values); Processed_Data_elbiologo.xlsx (Expression in RPKM for all detected transcripts)

Submission date

Nov 01, 2016

Last update date

May 15, 2019

Contact name

Miguel A Hernández-Prieto

Organization name

University of Sydney

Department

School of life and environmental sciences

Lab

ARC centre of excellence for translational photosynthesis