E-MTAB-5153_2
RNA-seq transcriptome profiling of the psychrophilic diatom Fragilariopsis cylindrus under six treatments simulating conditions within sea ice
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.
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.