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| Status |
Public on Aug 13, 2021 |
| Title |
Flight, met1-7, Roots, replicate 4 |
| Sample type |
SRA |
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| Source name |
solid 0.5x MS media plates; grown on the ISS, 11 days old plant harvested into KFT containing RNAlater fixative
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| Organism |
Arabidopsis thaliana |
| Characteristics |
cultivar: Col-0
genotype: met1-7
tissue: Roots
organism part: Whole roots from 11 day old seedling
age: 11 days old plants, 24hr light grown
biosource type: RNAlater fixed_sample
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| Treatment protocol |
On Orbit Operations and harvest: At 11 days, seedlings were harvested by an astronaut into KFT (Kennedy Fixation Tube) containing RNAlater solutions. Once the plants were placed in the KFTs, the KFT was actuated with RNAlater to preserve the sample. At 24 hours post-harvest, KFTs were then transferred to MELFI freezer. Following Dragon Capsule splashdown in the Pacific Ocean, the KFTs transferred to the Cold Stowage charter plane at the Long Beach Airport, placed into an insulated shipper with dry ice, and flown to Johnson Space Center (JSC). The KFTs were then transferred via FedEx ground to the Kennedy Space Center. The KFTs were removed from dry ice and transferred to a -80°C freezer. The PIs retrieved the plant samples from the KFTs and transferred the samples back to the University of Florida. The harvested material was used to compare the transcriptomes of each genotype. The patterns of gene expression and the genome-wide methylation profiles were compared between treatments (spaceflight versus ground control) and within each genotype.
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| Growth protocol |
The laboratory preparations and launch conditions: Dry, sterilized Arabidopsis seeds were planted aseptically on the surface of 10-cm2 solid media plates comprised of 0.5% Phytagel/0.5× MS media, and then immediately wrapped in light-tight black cloth (Duvetyne, SeattleFabrics.com) and transported to KSC (Kennedy Space Center), FL, USA. The wrapped plates were stored at temperatures between 4 and 10 °C until launch within a cold stowage bag nominally at 4 °C. The seeds remained dormant until removed from cold stowage and exposed to light at the initiation of the experiment on the ISS (International Space Station). Plant growth on the ISS and ISSES: The plates were installed into the Vegetable Production System (Veggie) hardware on the ISS and a comparable set installed into the Veggie hardware on the ISS Environmental Simulator (ISSES) chamber at Kennedy Space Center (KSC). These plates made up the NASA Advanced Plant Experiment 04 – Epigenetic Expression (APEX04-EPX) experiment that was launched on the SpaceX mission CRS-10. Plants in the Veggie hardware were exposed to constant light conditions of 100-135 µmoles/m2/s PAR and grown for eleven days (11d) before being harvested into Kennedy Space Center Fixation Tubes (KFTs) and fixed in RNAlater™ (Ambion, Grand Island, NY, USA). Samples from each plate were harvested into individual KFTs. The KFTs were then stowed at -80°C in the MELFI freezer aboard the ISS. The ground control plates were grown inside the Veggie ground unit within the ISSES (International Space Station Environment Simulator) chamber at KSC with the 48-h delay. The ISSES chamber replicated the temperature, CO2 levels and lighting that had been experienced by the spaceflight plants in the previous 48 h. Ground control plants were harevested after 11 days of growth into KFTs with RNAlater™ and stored in a at -80°C freezer.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Seedlings stored in RNA later were recovered from -80°C freezer. Each tube containing the seedlings was allowed to thaw in the fridge overnight. After completely thawing the samples to room temperature, the seedlings were observed under the microscope. The preserved seedlings from the spaceflight and ground control harvests were dissected into distinct plant parts: leaf, hypocotyl, and root. DNA extraction was done using a modified phenol/chloroform protocol (LeFrois et al., 2016) and genomic-wide bisulfite sequencing was performed using a similar procedure as described by (Wang et al., 2013). 700-1700 ng of genomic DNA (>5 Kb in length) observed on the TapeStation Genomic Screen Tape (Agilent) was processed for sequencing library construction.
Library preparation: DNA was transferred into 6x16 mm glass microtubes with AFA fiber and pre-slit snap caps (Cat# 520045, Covaris, Inc.) and sheared into average fragments size of ~400 bp, using the Covaris S220 ultrasonic disruptor. Short DNA fragments (<100 bp) were removed using AMPure magnetic beads (Cat# A63881, Beckman Coulter) at a 1:1 bead to sample ratio. 100-250 ng of clean, fragmented DNA was used for the Illumina sequencing library construction. The NEBNext® UltraTM II DNA Illumina construction kit (Cat# E7645S, NEB) in conjunction with the Illumina-specific methylated and dual-index barcoded adaptors (Cat# E7600S NEB) were used as described in the manufacturer’s guidelines. Illumina libraries (containing methylated adaptors) were subjected to sodium bisulfite treatment using the EZ DNA Methylation Direct kit (ZYMO Research, Cat #D5020) according to the manufacturer's instructions. The resulting libraries were enriched by a 13-15 cycle amplification using an uracil-insensitive polymerase (EpiMark hot start Taq polymerase, NEB, Cat#M0490S). The amplified library products were separated on a 2% agarose gel from which library fragments in the 250-500 bp range were excised (QIAquick gel extraction kit, Cat# 28704, QIAGEN) and AMPure purified (Cat# A63881, Beckman Coulter). Gel staining was done with SYBR safe (Life Technologies) and visualized on a blue light transilluminator (Life Technologies) to avoid UV damage to the DNA. The final libraries were quantitated by the QUBIT fluorometer, sized on the Agilent TapeStation (DNA5000 Screen Tape) and by qPCR with the Kapa SYBR Fast qPCR reagents (Cat# KK4824, Kapa Biosystems) with monitoring on an ABI7900HT real-time PCR system (LifeTechnologies). The average library size was 350 bp. Care was taken to generate WGBS libraries that were approximately the same size as the RNA-Seq libraries.
Illumina sequencing: Sequencing experiments were performed at the Interdisciplinary Center for Biotechnology Research (ICBR) gene expression and sequencing core, University of Florida. Uniquely barcoded libraries were normalized to 2.5 nM and pooled (equimolarly) for sequencing on the HiSeq3000 Illumina sequencer. Bisulfite-converted sequencing libraries were sequenced together with RNAseq libraries (uniquely barcoded) to maximize data output. The RNAseq libraries in the pool served to compensate for the low base diversity of bisulfite-converted genomic libraries. The final library was created by mixing RNA-seq vs bisulfite-converted libraries at a 60%:40% ratio, with a mere 1% PhiX spike-in. Library pools were processed according the Illumina protocol (HiSeq3000) for clustering on the cBOT machine. After denaturation, neutralization and mixing with the ExAmp reagent, the final pool concentration for clustering was 0.25 nM. Sequencing was done using a 2x101 cycles format (paired-end configuration). The 48-sample project was sequenced on 12 lanes for a robust reads/lane output.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
FER.vs.FMR.full.CG.txt
FER.vs.FMR.full.CHG.txt
FER.vs.FMR.full.CHH.txt
FER.vs.FMR.genes01.full.CG.txt
FER.vs.FMR.genes01.full.CHG.txt
FER.vs.FMR.genes01.full.CHH.txt
FER.vs.FMR.genes02.full.CG.txt
FER.vs.FMR.genes02.full.CHG.txt
FER.vs.FMR.genes02.full.CHH.txt
FER.vs.FMR.genes03.full.CG.txt
FER.vs.FMR.genes03.full.CHG.txt
FER.vs.FMR.genes03.full.CHH.txt
FER.vs.FMR.genes04.full.CG.txt
FER.vs.FMR.genes04.full.CHG.txt
FER.vs.FMR.genes04.full.CHH.txt
FER.vs.FMR.genes05.full.CG.txt
FER.vs.FMR.genes05.full.CHG.txt
FER.vs.FMR.genes05.full.CHH.txt
FMR.vs.FCR.full.CG.txt
FMR.vs.FCR.full.CHG.txt
FMR.vs.FCR.full.CHH.txt
FMR.vs.FCR.genes01.full.CG.txt
FMR.vs.FCR.genes01.full.CHG.txt
FMR.vs.FCR.genes01.full.CHH.txt
FMR.vs.FCR.genes02.full.CG.txt
FMR.vs.FCR.genes02.full.CHG.txt
FMR.vs.FCR.genes02.full.CHH.txt
FMR.vs.FCR.genes03.full.CG.txt
FMR.vs.FCR.genes03.full.CHG.txt
FMR.vs.FCR.genes03.full.CHH.txt
FMR.vs.FCR.genes04.full.CG.txt
FMR.vs.FCR.genes04.full.CHG.txt
FMR.vs.FCR.genes04.full.CHH.txt
FMR.vs.FCR.genes05.full.CG.txt
FMR.vs.FCR.genes05.full.CHG.txt
FMR.vs.FCR.genes05.full.CHH.txt
FMR.vs.GMR.full.CG.txt
FMR.vs.GMR.full.CHG.txt
FMR.vs.GMR.full.CHH.txt
FMR.vs.GMR.genes01.full.CG.txt
FMR.vs.GMR.genes01.full.CHG.txt
FMR.vs.GMR.genes01.full.CHH.txt
FMR.vs.GMR.genes02.full.CG.txt
FMR.vs.GMR.genes02.full.CHG.txt
FMR.vs.GMR.genes02.full.CHH.txt
FMR.vs.GMR.genes03.full.CG.txt
FMR.vs.GMR.genes03.full.CHG.txt
FMR.vs.GMR.genes03.full.CHH.txt
FMR.vs.GMR.genes04.full.CG.txt
FMR.vs.GMR.genes04.full.CHG.txt
FMR.vs.GMR.genes04.full.CHH.txt
FMR.vs.GMR.genes05.full.CG.txt
FMR.vs.GMR.genes05.full.CHG.txt
FMR.vs.GMR.genes05.full.CHH.txt
FMR-winavg.CG.txt
FMR-winavg.CHG.txt
FMR-winavg.CHH.txt
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| Data processing |
The short reads from the uniquely barcoded bisulfite-converted genomic libraries were trimmed using trimmomatic v 0.36 and quality control on the original and trimmed reads was performed using FastQC v 0.11.4 and MultiQC v 1.1. The bisulfite-converted reads were aligned to the TAIR10 genome using BSMAP. Methylation calling was performed with CSCALL and the differential methylation analysis was performed using the MCOMP program, which is part of the MOABS package.
A cytosine site that had at least a 10x read coverage in at least 2 out of the 4 replicates were included in the downstream analyses. Methylated cytosine sites for which the p-value of the difference between test and control methylation rates was below 0.01 were considered differentially methylated cytosines (DmCs). Differentially methylated regions were defined by comparing the average methylation levels within a 100 bp window between spaceflight and ground controls and those with statistical significance (p<0.01) were used in the analysis.
Genome_build: TAIR10
Supplementary_files_format_and_content: .csv files containing methylation levels in a 100 bp sliding window across the genome and differential methylation between the spaceflight vs. ground control comparisons.
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| Submission date |
Aug 13, 2018 |
| Last update date |
Aug 13, 2021 |
| Contact name |
Robert J. Ferl |
| E-mail(s) |
ferllabuf@gmail.com
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| Phone |
352-273-8030
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| Organization name |
University of Florida
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| Department |
Horticultural Sciences
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| Lab |
Ferl's lab
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| Street address |
1301 Fifield Hall PO Box 110690
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| City |
Gainesville |
| State/province |
Florida |
| ZIP/Postal code |
32611 |
| Country |
USA |
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| Platform ID |
GPL21179 |
| Series (2) |
| GSE118483 |
Characterizing Epigenetic Changes in Methylation Mutants (elp2-5 and met1-7) in Response to Spaceflight. [Bisulfite-Seq] |
| GSE118503 |
Characterizing Epigenetic Changes in Methylation Mutants (elp2-5 and met1-7) in Response to Spaceflight. |
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| Relations |
| BioSample |
SAMN09814151 |
| SRA |
SRX4549379 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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