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| Status |
Public on Aug 13, 2021 |
| Title |
elp2-5, Roots, Ground control, replicate 4 [ERG4] |
| Sample type |
SRA |
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| Source name |
solid 0.5x MS media plates; grown on the ground, 11 days old plant harvested into KFT containing RNAlater fixative
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| Organism |
Arabidopsis thaliana |
| Characteristics |
cultivar: Col-0
genotype: elp2-5
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 |
total RNA |
| 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. Total RNA extraction: Total RNA was extracted using Qiashredder and RNAeasy™ kits from QIAGEN (QIAGEN Sciences, MD, USA) according to the manufacturer’s instructions. Residual DNA was removed by performing an on-column digestion using an RNase Free DNase (QIAGEN GmbH, Hilden, Germany). Integrity of the RNA was evaluated using the Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA).
Library preparation: RNAseq library were constructed using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations. Basically, 2ul of of 1:200 diluted RNA spike-in ERCC (half amount of suggested in the ERCC user guide: Cat# 4456740) spike to 1000 ng of total RNA followed by mRNA isolating using NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs, catalog # E7490). Then followed by RNA library construction with NEBNext Ultra Directional Lib Prep (New England Biolabs, catalog #E7420) according to the manufacturer's user guide. Briefly, RNA is fragmented in NEBNext First Strand Synthesis Buffer by heating at 94 °C for desired time. This step is followed by first strand cDNA synthesis using reverse transcriptase and oligodT primers. Synthesis of ds cDNA is performed using the 2nd strand master mix provided in the kit, followed by end-repair and adaptor ligation. At this point, Illumina adaptors are ligated to the sample. Finally, library is enriched (each library has a unique barcode) by 10 cycles of amplification, and purified by Agencourt AMPure beads (Beckman Coulter, catalog # A63881). 48 barcoded libraries were sized on the bioanalyzer, quantitated by QUBIT. Finally, 48 individual libraries were pooled with equimolar.
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 |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
diff_expr_genes_root_direct_11_07_2017.xlsx
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| Data processing |
The quality of the RNA-Seq sequence data was first evaluated using FastQC prior to further downstream analysis. Low quality sequences were removed and poor quality part of the reads were trimmed using Trimmomatic. Star Aligner was used to map high quality paired-end reads to TAIR10 genome. Gene expression was obtained using RSEM. The expected read counts and Fragments Per Kilobase of transcript per Million mapped reads (FPKM), were extracted for further analysis. A generalized linear regression model was built to perform the differential gene analysis using edgeR. Prior to the differential analysis, hierarchical clustering and Principal Component Analysis (PCA) were conducted to identify the potential outlier of the samples. The thresholds for calling significantly differential expressed genes were set at FDR 0.05, the fold change of greater than 2, and the average FPKM for at least one of each comparison group is higher than 0.
Genome_build: TAIR10
Supplementary_files_format_and_content: .xls spreadsheets containing lists of genes, FPKM values, and CPM values.
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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) |
| GSE118502 |
Characterizing Epigenetic Changes in Methylation Mutants (elp2-5 and met1-7) in Response to Spaceflight. [RNA-Seq] |
| GSE118503 |
Characterizing Epigenetic Changes in Methylation Mutants (elp2-5 and met1-7) in Response to Spaceflight. |
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| Relations |
| BioSample |
SAMN09836182 |
| SRA |
SRX4553600 |
| 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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