RNA-seq transcriptome profiling of the psychrophilic diatom Fragilariopsis cylindrus under six treatments simulating conditions within sea ice
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ABSTRACT:
PROVIDER: PRJEB23123 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR2178840_1.fastq.gz | Fastqsanger.gz | |||
| ERR2178840_2.fastq.gz | Fastqsanger.gz | |||
| ERR2178850_1.fastq.gz | Fastqsanger.gz | |||
| ERR2178850_2.fastq.gz | Fastqsanger.gz | |||
| ERR2178841_1.fastq.gz | Fastqsanger.gz |
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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 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: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:Disentangling the genomic complexity of the Fragilariopsis cylindrus (CCMP1102) genome.
| PRJEB51975 | ENA
Project description:RNA-seq transcriptome profiling of the psychrophilic phytoplankton Fragilariopsis cylindrus under environmentally relevant growth limitations
| PRJEB15139 | ENA
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:The Southern Ocean diatom Fragilariopsis cylindrus was grown under half-saturating concentrations of silicate (0.3 uM) and in the blue (480 - 540 nm) and red (550 - 700 nm) light spectrum to investigate gene expression responses using RNA-seq relative to optimal growth conditions (deposited under accession E-MTAB-5024). RNA-seq was performed on three replicate samples for each experimental condition. 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.
2020-12-31 | E-MTAB-5429 | biostudies-arrayexpress
Project description:RNA-seq transcriptome profiling of the psychrophilic diatom Fragilariopsis cylindrus grown at 0 degree Celsius and two levels of salinity
| PRJEB36182 | ENA
Project description:The genome of the cold-adapted diatom Fragilariopsis cylindrus is characterized by highly diverged haplotypes that intersperse its homozygous genome. Here, we describe how a combination of PacBio DNA and Illumina RNA sequencing can be used to resolve this complex genomic landscape locally into the highly diverged haplotypes, and how to map various environmentally controlled transcripts onto individual haplotypes. We assembled PacBio sequence data with the FALCON assembler and created a haplotype resolved annotation of the assembly using annotations of a Sanger sequenced F. cylindrus genome. RNA-seq datasets from six different growth conditions were used to resolve allele-specifc gene expression in F. cylindrus. This approach enables to study differential expression of alleles in a complex genomic landscape and provides a useful tool to study how diverged haplotypes in diploid organisms are used for adaptation and evolution to highly variable environments.
| S-EPMC5634323 | biostudies-literature