RNA-seq transcriptome profiling of the Southern Ocean diatom Fragilariopsis cylindrus under different experimental conditions, silica limitation and two different light spectra
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2000
ORGANISM(S): Fragilariopsis cylindrus
SUBMITTER: Jan Strauss
PROVIDER: E-MTAB-5429 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:We performed RNA-sequencing experiments to examine the differential regulation of genes in the genome of the Southern Ocean diatom Fragilariopsis cylindrus including diverged alleles. RNA-seq was performed on three replicate samples for each experimental condition. Phytoplankton cells were grown under six different experimental conditions including (1) optimal growth, (2) freezing temperatures, (3) elevated temperature, (4) elevated carbon dioxide concentrations, (5) low iron concentrations and (6) prolonged darkness. Total RNA was extracted using a guanidinium thiocyanate-phenol-chloroform extraction protocol, followed by DNase I treatment and RNA purification (Quiagen). First strand cDNA synthesis was performed using random hexamers. Library preparation was performed using the RNA-seq Sample Prep Kit (Illumina) and sequencing was conducted according to the TruSeq RNA sequencing protocol (Illumina) All samples were sequenced together in one flowcell on one lane.
2016-12-16 | E-MTAB-5024 | biostudies-arrayexpress
Project description:Background: Marine phytoplankton are responsible for 50% of the CO2 that is fixed annually worldwide and contribute massively to other biogeochemical cycles in the oceans. Diatoms and coccolithophores play a significant role as the base of the marine food web and they sequester carbon due to their ability to form blooms and to biomineralise. To discover the presence and regulation of short non-coding RNAs (sRNAs) in these two important phytoplankton groups, we sequenced short RNA transcriptomes of two diatom species (Thalassiosira pseudonana, Fragilariopsis cylindrus) and validated them by Northern blots along with the coccolithophore Emiliania huxleyi. Results: Despite an exhaustive search, we did not find canonical miRNAs in diatoms. The most prominent classes of sRNAs in diatoms were repeat-associated sRNAs and tRNA-derived sRNAs. The latter were also present in E. huxleyi. tRNA-derived sRNAs in diatoms were induced under important environmental stress conditions (iron and silicate limitation, oxidative stress, alkaline pH), and they were very abundant especially in the polar diatom F. cylindrus (20.7% of all sRNAs) even under optimal growth conditions. Conclusions: This study provides first experimental evidence for the existence of short non-coding RNAs in marine microalgae. Our data suggest that canonical miRNAs are absent from diatoms. However, the group of tRNA-derived sRNAs seems to be very prominent in diatoms and coccolithophores and may be used for acclimation to environmental conditions.
2015-03-06 | GSE57987 | GEO
Project description:Background: Marine phytoplankton are responsible for 50% of the CO2 that is fixed annually worldwide and contribute massively to other biogeochemical cycles in the oceans. Diatoms and coccolithophores play a significant role as the base of the marine food web and they sequester carbon due to their ability to form blooms and to biomineralise. To discover the presence and regulation of short non-coding RNAs (sRNAs) in these two important phytoplankton groups, we sequenced short RNA transcriptomes of two diatom species (Thalassiosira pseudonana, Fragilariopsis cylindrus) and validated them by Northern blots along with the coccolithophore Emiliania huxleyi. Results: Despite an exhaustive search, we did not find canonical miRNAs in diatoms. The most prominent classes of sRNAs in diatoms were repeat-associated sRNAs and tRNA-derived sRNAs. The latter were also present in E. huxleyi. tRNA-derived sRNAs in diatoms were induced under important environmental stress conditions (iron and silicate limitation, oxidative stress, alkaline pH), and they were very abundant especially in the polar diatom F. cylindrus (20.7% of all sRNAs) even under optimal growth conditions. Conclusions: This study provides first experimental evidence for the existence of short non-coding RNAs in marine microalgae. Our data suggest that canonical miRNAs are absent from diatoms. However, the group of tRNA-derived sRNAs seems to be very prominent in diatoms and coccolithophores and may be used for acclimation to environmental conditions. RNA-seq study of sRNA populations in two species of diatoms using Illumina GAII high-throughput sequencing
2015-03-06 | E-GEOD-57987 | biostudies-arrayexpress
Project description:The sea-ice dwelling diatom Fragilariopsis Cylindrus was cultured for 4 months under dark or light exposed conditions to mimic the effects of Antarctic winter growth conditions. Cells were harvested periodically and LFQ proteomics used to investigate the molecular mechanisms of dark survival.
2019-04-23 | PXD007098 | Pride
Project description:RNA-seq transcriptome profiling of the Southern Ocean diatom Fragilariopsis cylindrus under different experimental conditions, silica limitation and two different light spectra
| PRJEB19093 | ENA
Project description:We performed RNA-sequencing experiments to examine gene expression in the genome of the cold-adapted (psychrophilic) marine diatom Fragilariopsis cylindrus to six different treatments simulating conditions found within sea ice. RNA-seq was performed on replicate samples for each experimental condition. Phytoplankton cells were grown under six different experimental conditions including (I) 0℃ and salinity 34 (seawater before ice formation), (II) two days at -4℃ and salinity 34 (early freezing or frazil ice formation), (III) eight days at -4℃ and salinity 34 (late freezing, frazil ice layer formation), (IV) two days at -4℃ and salinity 52 (initialization of brine channel formation in ice), (V) eight days at -4℃ and salinity 52 (established brine channel formation and young consolidated columnar ice), and (VI) two days at -8℃ and salinity 52 (further temperature stress). Total RNA was extracted using a guanidinium thiocyanate-phenol-chloroform extraction protocol, followed by DNase I treatment and RNA purification (Quiagen). First strand cDNA synthesis was performed using random hexamers. Library preparation was performed using the RNA-seq Sample Prep Kit (Illumina) and sequencing was conducted according to the TruSeq RNA sequencing protocol (Illumina). Illumina TruSeq RNA library production process: The libraries for this project were constructed with the PerkinElmer Sciclone using the TruSeq RNA protocol (Illumina 15026495 Rev.B). The library preparation involved an initial QC of the RNA using Qubit DNA (Life technologies Q32854) and RNA (Life technologies Q32852) assays as well as a quality check using the Bioanalyzer with the Nano kit (Agilent 5067-1511). 1ug of this RNA was purified to extract mRNA with a poly- A pull down using biotin beads and fragmented, first strand cDNA was synthesised followed by the second strand. The ends of the samples were repaired using the 3' to 5' exonuclease activity to remove the 3' overhangs and the polymerase activity to fill in the 5' overhangs creating blunt ends. A single ‘A’ nucleotide was added to the 3’ ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. A corresponding single ‘T’ nucleotide on the 3’ end of the adapter provided a complementary overhang for ligating the adapter to the fragment. This strategy ensured a low rate of chimera formation. The ligation of a number indexing adapters to the ends of the DNA fragments prepared them for hybridisation onto a flow cell. The ligated products were subjected to a bead based size selection using Beckman Coulter XP beads (Beckman Coulter A63880). This removed the majority of un-ligated adapters, as well as any adapters that may have ligated to one another. Prior to hybridisation to the flow cell the samples were PCR’d to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. The PCR was performed with a PCR primer cocktail that annealed to the ends of the adapter. The insert size of the libraries was verified by running an aliquot of the DNA library on a PerkinElmer GX using the High Sensitivity DNA chip (PerkinElmer CLS760672) and the concentration was determined by using a High Sensitivity Qubit assay and q-PCR. Illumina HiSeq 2000 clustering and sequencing: The 24 libraries were normalised and equimolar pooled to 10 nM using elution buffer (Qiagen). Each library pool was then diluted to 2 nM with NaOH and 5μL transferred into 995μL HT1 (Illumina) to give a final concentration of 10pM. 120 μL of each diluted library pool was then transferred into a 200 μL strip tube, spiked with 1% PhiX Control v3 and placed on ice before loading onto the Illumina cBot. The flow cell was clustered using TruSeq Paired End Cluster Generation Kit v3, following the Illumina PE_amplification_Linearization_Blocking_PrimerHyb recipe. Following the clustering procedure, the flow cell was loaded onto the Illumina HiSeq2000 instrument following the manufacturer’s instructions. The sequencing chemistry used was TruSeq SBS Kit v3-HS using HiSeq Control Software 1.4.8 and RTA 1.12.4.2. Each library pool was run in a single lane (lane 5 and 6) for 50cycles of each paired end read. Reads in bcl format were demultiplexed based on the 6 bp Illumina index by CASSAVA, allowing for a one base-pair mismatch per library, and converted to FASTQ format by bcl2fastq.
2018-01-18 | E-MTAB-5153 | biostudies-arrayexpress
Project description:The data set described here complements data available under accession number E-MTAB-5153. We performed bulk RNA-sequencing experiments to examine gene expression in the genome of the cold-adapted (psychrophilic) marine diatom Fragilariopsis cylindrus growing at 0 degree Celsius and salinity of 34 (normal seawater) and 52 (high salinity) to provide references (controls) to samples grown under a step-wise set of temperature reductions as characteristic during sea ice formation. RNA-seq was performed on replicate samples (N=3) for each experimental condition. Phytoplankton cells were grown under three different experimental conditions including (I) 20 days at 0℃ and salinity 34, (II) 14 days at 0℃ and salinity 52, and (III) 14 days at 0℃ and salinity 52. Samples were processed as described for experiment with accession number E-MTAB-5153. Total RNA was extracted using a guanidinium thiocyanate-phenol-chloroform extraction protocol, followed by DNase I treatment and RNA purification (Quiagen). First strand cDNA synthesis was performed using random hexamers. Library preparation was performed using the RNA-seq Sample Prep Kit (Illumina) and sequencing was conducted according to the TruSeq RNA sequencing protocol (Illumina). Illumina TruSeq RNA library production process: The libraries for this project were constructed with the PerkinElmer Sciclone using the TruSeq RNA protocol (Illumina 15026495 Rev.B). The library preparation involved an initial QC of the RNA using Qubit DNA (Life technologies Q32854) and RNA (Life technologies Q32852) assays as well as a quality check using the Bioanalyzer with the Nano kit (Agilent 5067-1511). 1ug of this RNA was purified to extract mRNA with a poly- A pull down using biotin beads and fragmented, first strand cDNA was synthesised followed by the second strand. The ends of the samples were repaired using the 3' to 5' exonuclease activity to remove the 3' overhangs and the polymerase activity to fill in the 5' overhangs creating blunt ends. A single ‘A’ nucleotide was added to the 3’ ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. A corresponding single ‘T’ nucleotide on the 3’ end of the adapter provided a complementary overhang for ligating the adapter to the fragment. This strategy ensured a low rate of chimera formation. The ligation of a number indexing adapters to the ends of the DNA fragments prepared them for hybridisation onto a flow cell. The ligated products were subjected to a bead based size selection using Beckman Coulter XP beads (Beckman Coulter A63880). This removed the majority of un-ligated adapters, as well as any adapters that may have ligated to one another. Prior to hybridisation to the flow cell the samples were PCR’d to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. The PCR was performed with a PCR primer cocktail that annealed to the ends of the adapter. The insert size of the libraries was verified by running an aliquot of the DNA library on a PerkinElmer GX using the High Sensitivity DNA chip (PerkinElmer CLS760672) and the concentration was determined by using a High Sensitivity Qubit assay and q-PCR. Illumina HiSeq 2000 clustering and sequencing: The 24 libraries were normalised and equimolar pooled to 10 nM using elution buffer (Qiagen). Each library pool was then diluted to 2 nM with NaOH and 5μL transferred into 995μL HT1 (Illumina) to give a final concentration of 10pM. 120 μL of each diluted library pool was then transferred into a 200 μL strip tube, spiked with 1% PhiX Control v3 and placed on ice before loading onto the Illumina cBot. The flow cell was clustered using TruSeq Paired End Cluster Generation Kit v3, following the Illumina PE_amplification_Linearization_Blocking_PrimerHyb recipe. Following the clustering procedure, the flow cell was loaded onto the Illumina HiSeq2000 instrument following the manufacturer’s instructions. The sequencing chemistry used was TruSeq SBS Kit v3-HS using HiSeq Control Software 1.4.8 and RTA 1.12.4.2. Each library pool was run in a single lane (lane 5 and 6) for 50cycles of each paired end read. Reads in bcl format were demultiplexed based on the 6 bp Illumina index by CASSAVA, allowing for a one base-pair mismatch per library, and converted to FASTQ format by bcl2fastq.
2022-04-30 | E-MTAB-8665 | biostudies-arrayexpress
Project description:Here, we examined the ramifications of between-species diversity by documenting the transcriptional response of three marine diatoms - Thalassiosira pseudonana, Fragilariopsis cylindrus, and Pseudo-nitzschia multiseries - to the onset of nitrate limitation of growth, a common limiting nutrient in the ocean. Less than 5% of orthologous genes, shared across the three diatoms, displayed the same transcriptional responses across species when growth was limited by nitrate availability. Orthologs, such as those involved in nitrogen uptake and assimilation, as well as carbon metabolism, were differently expressed across the three species. The two pennate diatoms, F. cylindrus and P. multiseries, shared 3,839 clusters without orthologs in the genome of the centric diatom T. pseudonana. A majority of these pennate-clustered genes, as well as the non-orthologous genes in each species, had minimal annotation information, but were often significantly differentially expressed under nitrate limitation, indicating their potential importance in the response to nitrogen availability. Despite these variations in the specific transcriptional response of each diatom, overall transcriptional patterns suggested that all three diatoms displayed a common physiological response to nitrate limitation that consisted of a general reduction in carbon fixation and carbohydrate and fatty acid metabolism and an increase in nitrogen recycling.
2014-03-25 | GSE56132 | GEO
Project description:Disentangling the genomic complexity of the Fragilariopsis cylindrus (CCMP1102) genome.
| PRJEB51975 | ENA