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.
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.
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 184.108.40.206. 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.
Project description:Mechanism, inheritance patterns and biological significance remain unclear. Here, we generated genome-wide integrated maps of H3K27me3 modification and transcriptome for Col, C24 and their F1 hybrid, and their clf mutants. We found that H3K27me3 modification profiles were divergent between Col and C24, and were inherited mainly additively in hybrid. By comparing H3K27me3 modification ratio between parents to allelic H3K27me3 modification ratio in hybrid, we identified cis- and trans-regulatory divergence for H3K27me3 modification variation between Col and C24, and found that cis-regulatory divergence was the predominant contributor to H3K27me3 variation. The majority of genes with both cis- and trans-regulatory divergence displayed "cis-trans" interaction, whereas "cis+trans" interaction resulted in higher magnitude of H3K27me3 variation. H3K27me3 modification variation was negatively correlated with gene expression variation between Col and C24, both of which might rise from the same cis-regulatory divergence. Moreover, cis-regulatory divergence could lead to the concurrent allelic H3K27me3 modification bias and allelic gene expression bias for auxin metabolic genes and stimulus responsive genes in Col×C24 hybrid. Natural variation of H3K27me3 modification and gene expression were changed upon CLF mutation, and heterosis, especially best-parent hetereosis, was largely compromised in clf hybrid. Together, our study provided a comprehensive analysis of regulatory divergence for natural variation of histone modification and its association with differential gene expression between Arabidopsis accessions and growth vigor in hybrid. Overall design: Genome-wide integrated maps and allelic expression of mRNA and histone modifications (H3K27me3) in the shoots of 14 day old seedlings of two Arabidopsis(Col and C24) and their hybrids (Col x C24).
Project description:Allelic differences between the two sets of chromosomes can affect the propensity of inheritance in humans, but the extent of such differences in the human genome has yet to be fully explored. Here, we delineate allelic chromatin modifications and transcriptomes amongst a broad set of human tissues, enabled by a chromosome-span haplotype reconstruction strategy1. The resulting haplotype-resolved epigenomic maps reveal extensive allele bias in the transcription of human genes as well as chromatin state, allowing us to infer cis-regulatory relationships between genes and their control sequences. These maps also uncover a new class of cis regulatory elements and detail activities of repetitive elements in various human tissues. The rich datasets described here will enhance our understanding of the mechanisms controlling tissue-specific gene expression programs. One replicate of Hi-C experiment in four human tissues with four different individuals (Thymus STL001, Aorta STL002, Leftventricle STL003, and Liver STL011).
Project description:ChIP-sequencing for H3K4me1, H3K4me3, and H3K27ac was performed in primary human hepatocytes from three individuals heterozygous for the T2D risk haplotype at the SLC16A11 locus. The goal was to identify allelic skews in chromatin marks at variants at the SLC16A11 locus. Overall design: Examination of 3 different histone modifications in 1 primary human cell type obtained from 3 individuals.