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Sample GSM2050608

Query DataSets for GSM2050608

Status

Public on Jan 30, 2017

Title

0.04 nM Fe'_rplA

Sample type

SRA

Source name

cells

Organism

Picosynechococcus sp. PCC 7002

Characteristics

strain: PCC7002 (BMB04)
developmental stage: exponential growth phase
label in the processed file: X4_2_A
sample type: Fe limited

Treatment protocol

Sample labels starting with 1_06_A, 2_06_B and 3_06_C, respectively represent the biological replicated of the iron (Fe) treatment with 0.6 nM of total Fe, which is equivalent to 0.003 nM of dissolved inorganic Fe (Severe Fe limitation). Sample labels starting with 4_2_A, 5_2_B and 6_2_C, respectively represent the biological replicated of the Fe treatment with 2 nM of total Fe, which is equivalent to 0.04 nM of dissolved inorganic Fe (Strong Fe limitation). Sample labels starting with 7_20_A, 8_20_B and 9_20_C, respectively represent the biological replicated of the Fe treatment with 20 nM of total Fe, which is equivalent to 0.41 nM of dissolved inorganic Fe (Mild Fe limitation). Sample labels starting with 10_200_A, 11_200_B and 12_200_C, respectively represent the biological replicated of the Fe treatment with 200 nM of total Fe, which is equivalent to 10.9 nM of dissolved inorganic Fe (Fe replete=control).

Growth protocol

Cyanobacterium Synechococcus sp. PCC7002, bioreporter BMB04, grown in Aquil culture medium amended with iron (Fe)-EDTA to maintain Fe concentration constant during the incubation time and achieve desired concentrations of dissolved inorganic Fe (Fe’) (10.9, 0.41, 0.04 and 0.003 nM). Cultures acclimated to grow under the selected Fe’ concentrations for at least 22 generations in diluted batches (<8 × 106 cell ml-1) were incubated in three biological replicates (inoculated with a biomass equivalent to 0.5 mg Chla l-1) in 2 l PC bottles for physiological and genomic analysis, at 22°C, with 50 μmol quanta m-2 s-1 (cool white fluorescent tubes) under a 12:12 light:dark cycle.

Extracted molecule

total RNA

Extraction protocol

Cells for RNA extraction were harvested in mid-exponential growth phase by filtration through 0.4 mm polycarbonate membrane and cemtrifugated at 6000 × g for 8 min at 4 ºC. The pellets were snap frozen in liquid nitrogen. RNA was isolated using TRI Reagent (Sigma-Aldrich, Buch, Switzerland) following provider’s instructions. Ribosomal RNA (rRNA) was depleted using Ribo-Zero™ rRNA Removal Kit using gram-negative bacteria-specific capture oligonucleotides (Epicentre, Madison, WI, USA). RNA purity and integrity was determined using the Bioanalyzer RNA 6000 Nano Kit (Agilent Technologies, Inc., Santa Clara, CA, USA).
Libraries were prepared using 200 ng of RNA with the Illumina TruSeq Stranded mRNA kit and sequenced generating 100 bp single reads on an Illumina HiSeq 2500 System (Illumina, San Diego, CA, USA).
Quality control was performed using FastQC tool (Babraham Bioinformatics, UK). Reads were aligned to the genome of Synechococcus sp. PCC7002 build from the Cyanobase (Fujisawa et al., 2013) and further analyzed using TopHat 2.0 mapper software (Trapnell et al., 2009). Table of counts were obtained using the HTSeq 0.5.3.p9 (Anders et al., 2015). Reads mapped to the reference genome at 88.7 ±6.9 %.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Quality control was performed using FastQC tool (Babraham Bioinformatics, UK)
Mapping was performed using TopHat 2.0 mapper software (Trapnell et al., 2009)
Table of counts were obtained using the HTSeq 0.5.3.p9 (Anders et al., 2015)
Genome_build: Reads were aligned to the genome of Synechococcus sp. PCC7002 build from the Cyanobase (Fujisawa et al., 2013)
Supplementary_files_format_and_content: Normalized_counts_cpm_replicates.txt

Submission date

Jan 28, 2016

Last update date

May 15, 2019

Contact name

Sonia Blanco-Ameijeiras

Organization name

University of Geneva

Department

F.-A. Forel Institute

Lab

Marine and Lake Biogeochemistry