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Sample GSM1872159

Query DataSets for GSM1872159

Status

Public on Jun 08, 2016

Title

SM_day 9_27C_sample 3

Sample type

SRA

Source name

SM_day 9_27C

Organism

Cladocopium sp. C1

Characteristics

population: South Molle (SM)
population source: South Molle Island, Great Barrier Reef
thermal tolerance phenotype: sensitive
sampling time point: day 9
temperature treatment: 27C
incubator: incubator 2
sample type: cell culture
medium: filtered sea water + IMK nutrient supplement
coral host from which it was isolated: Acropora tenuis

Treatment protocol

Two flasks per population were randomly assigned to each of four experimental incubators and acclimated at 27°C for two weeks. Two incubators were ramped on day 0 at 0.5°C/h to 32°C for the heat stress temperature treatment, while two incubators remained at 27°C.

Growth protocol

Symbiodinium populations were cultured in filtered seawater supplemented with Daigo IMK (Wako Pure Chemical Industries, Ltd.). For temperature experiments, 5x10^7 cells (1.5x10^6 cells/ml) of each population were added to eight replicate culture flasks per population. Light was provided at an intensity of 30 µmol quanta m-2 s-1 with a 12 h light-dark cycle.

Extracted molecule

polyA RNA

Extraction protocol

Precisely after six hours of light exposure, 2-4 x 10^6 cells per population were immediately snap frozen in liquid nitrogen within 10 s of removal from experimental incubators and stored at -80°C. Snap frozen cells were thawed at room temperature and pelleted at 4°C (3,000 x g, 5 min). Media was removed, and pellets were lysed in buffer RLT (RNeasy plant mini kit, Qiagen) containing β-mercaptoethanol by bead beating with 0.3 g of 710-1,180 µm acid-washed glass beads (Sigma) using a TissueLyser II (Qiagen) for 90 s at 30 Hz. RNA was extracted and purified using the RNeasy plant mini kit (Qiagen) with an added on-column DNase I treatment (Qiagen).
Total RNA (150-500 ng) of each sample was sent to the Australian Genome Research Facility for confirmation of high quality RNA on an Agilent 2100 bioanalyzer, polyA-purification, Illumina TruSeq stranded library preparation, and sequencing on an Illumina HiSeq2500 (single end 100 bp, ~107 reads per sample)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

processed data file: day9_SM_RSEM_genes.counts.matrix

Data processing

Illumina Truseq (TruSeq3-SE) adapters were removed from sequence reads using Trimmomatic.
Prinseq was used to remove poly-A tails (min 6-A tail) and to filter short (min length 60 bases), low quality (min mean quality score 20, 4 base window, 1 base step), and low complexity sequences (dust method threshold 7).
RNA sequences from the 24 samples per population (four replicates, two temperature treatments, three time points) were combined for de novo assembly of the SM and MI transcriptomes using Trinity (version: 2.0.6).
transcripts shorter than 250 bp were removed from the assembeled transcriptomes
redundant transcripts (similarity > 90%) were removed from the assembled transcriptomes using cd-hit-est
Genome_build: the assembeled transcriptome
Supplementary_files_format_and_content: tab-delimited text files include expression count matrices for each sample at each time point

Submission date

Sep 08, 2015

Last update date

May 15, 2019

Contact name

Rachel Levin

E-mail(s)

[email protected]

Organization name

University of New South Wales

Street address

UNSW