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Sample GSM2519317 Query DataSets for GSM2519317
Status Public on Mar 01, 2020
Title Ground Control, 8 days old, Sku5 Rep2
Sample type SRA
Source name solid 0.5x MS media plates; grown on the ground, 8 days old plant harvested into KFT containing RNAlater fixative
Organism Arabidopsis thaliana
Characteristics cultivar: Sku5
material type: Roots
organism part: whole root of the 8 days old seedling
age: 8 days old plants, light grown
biosource type: RNAlater fixed_sample
Treatment protocol On Orbit Operations and harvest: At four or for the second experimental set at eight 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 was compared between treatments (spaceflight versus ground control) within each genotype of same age or between two ages within same genotype and within same treatment.
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, 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 APEX03-2 experiment plates were grown in the Vegetable Production System (VPS/Veggie) hardware on the Columbus Module of the ISS. The 10-cm2 Petri plates were housed perpendicular (“vertical”) to the overhead LED lighting of the VPS. Plants received between 100–135 μmoles/m2/s PAR inside the VPS. Four and eight days after germination was initiated by unwrapping the plates and exposing them to light in the VPS. The ground control plates were grown inside the VPS 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.
Extracted molecule total RNA
Extraction protocol Sample preparation for transcriptomics: Seedlings stored in RNAlater 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. Roots of the preserved seedlings from the spaceflight and ground control harvests were dissected and the rest of the seedlings were saved in RNAlater and stored back to -80°C freezer. For 4 day old roots 5-8 roots were combined to serve as one biological replica. For 8 day old roots 2-3 roots were used to form one biological replica. In each case the four biological replicas were used for the transcriptomic analysis. 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 Libraries Preparation using SMART-Seq V4 ultra low input RNA kit for sequencing combined with Illumina Nextera DNA Sample Preparation Kit Ten ng of total RNA was used for cDNA library construction using CloneTech SMART-Seq V4 ultra low input RNA kit for sequencing (Clontech Laboratories, Inc, cat#: 634890) according to manufacturer's protocol. Briefly, 1st strand cDNA was primed by the SMART-Seq v4 oligonucleotide and then base-pairs with these additional nucleotides, creating an extended template. The reverse transcriptase then switches templates and continues transcribing to the end of the oligonucleotide, resulting full-length cDNA that contains an anchor sequence that serves as a universal priming site for second strand synthesis. cDNA was amplified with primer II A for 8 PCR cycles. Then Illumina sequencing libraries were generated with 150 pg of cDNA using Illumina Nextera DNA Sample Preparation Kit (Cat#: FC-131-1024) according to manufacturer’s instructions. Briefly, 150 pg of cDNA was fragmented by tagementation reaction and then adapter sequences added onto template cDNA by PCR amplification. Libraries were quantitated by Bioanalyzer and qPCR (Kapa Biosystems, catalog number: KK4824). Finally, the libraries were pooled equal molar concentration and sequenced by Illumina 2X75 NextSeq 500.
Illumina sequencing: Sequencing experiments were performed at the Interdisciplinary Center for Biotechnology Research (ICBR) gene expression and sequencing core, University of Florida. In preparation for sequencing, barcoded libraries were sized on the bioanalyzer, quantitated by QUBIT and qPCR (Kapa Biosystems, catalog number: KK4824). Individual samples were pooled equimolarly at 4 nM. This “working pool” was used as input in the NextSeq500 instrument sample preparation protocol (Illumina, Part # 15048776, Rev A). Typically, a 1.3 pM library concentration resulted in optimum clustering density in our instrument (i.e., ~200,000 clusters per mm2). Samples were sequenced on 5 flowcells (5 NextSeq 500 runs), using a 2x75 cycles (paired-end) configuration. A typical sequencing run in the NextSeq500 produced 750-800 million paired-end read with a Q30>=85%. For RNA seq, around 40 million reads provided sufficient depth for transcriptome analysis.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description FLT 8d Sku5 to GC 8d Sku5
GC 4d Sku5 to GC 8d Sku5
GC 8d Sku5 to GC 8d WS
Data processing The data analysis was performed at the ICBR bioinformatics core, University of Florida. Input fastq files were trimmed to remove the adapters and low-quality bases using trimmomatic (version 0.36) with the following parameters: LEADING:3, TRAILING:3, SLIDINGWINDOW:4:15, MINLEN:50. Quality control on the trimmed reads was performed using FastQC version 0.11.2. Reads were aligned to the WS genome (Gan et al. Nature. 2011 Aug 28; 477(7365):419-23.) using the STAR aligner (version 2.5.1b), and duplicate reads due to PCR artifacts were removed using the Picard MarkDuplicates tool. In total, 502 million reads were aligned to the transcriptome.
Expression quantification and differential gene expression analysis were performed using cufflinks and cuffdiff respectively (version 2.2.1) with default parameters. The cuffdiff output files were parsed with custom scripts to generate the final annotated tables of differentially expressed genes.
Submission date Mar 02, 2017
Last update date Mar 01, 2020
Contact name Robert J. Ferl
Phone 352-273-8030
Organization name University of Florida
Department Horticultural Sciences
Lab Ferl's lab
Street address 1301 Fifield Hall PO Box 110690
City Gainesville
State/province Florida
ZIP/Postal code 32611
Country USA
Platform ID GPL19580
Series (1)
GSE95620 During development, the Sku5 mutant roots engage different genes than wild type WS roots, either on the ground or in spaceflight.
BioSample SAMN06468825
SRA SRX2609542

Supplementary data files not provided
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Raw data are available in SRA
Processed data are available on Series record

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