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SRX2589023: GSM2509423: Ground Control, 4 days old, WS Rep1; Arabidopsis thaliana; RNA-Seq
20 ILLUMINA (NextSeq 500) runs: 20.3M spots, 2.9G bases, 1.2Gb downloads

Submitted by: NCBI (GEO)
Study: Plant development on ISS differes from the development on the ground and is influenced by the genetic background.
show Abstracthide Abstract
The development of Col-0 and WS plants on orbit differs from that on the ground as demonstrated by the comparison of the gene expression profiles between 4 days old to 8 days old plant in the two environments. The Col-0 plants used different genes reflecting different physiological processes than WS plants, suggesting the role of the genetic background in the developmental decisions. The 4 days old Col-0 plant in orbit showed deficit in wax and suberin production relatively to the 8 days old plants, the difference unregistered on the ground. There was more dramatic difference in the overexpression of the root system development and anatomical structure development genes in 8 days old plants than in 4 days old plant in both genotypes on the ground than in flight, implying smaller root developmental gap between 4 days and 8 days old roots in orbit. The WS plant uniquely showed overexpression of the photosynthesis related genes in the 4 days old roots relative to 8 days old root in the spaceflight environment but not on the ground. The seeds germinated in the novel growth environment of ISS implemented different developmental strategies as captured by the genes expression patterns, than seeds developing on the ground. Overall design: APEX03-2 (Advanced Plant Experiment 03-2) also identified as TAGES-Isa (Transgenic Arabidopsis Gene Expression System—Intracellular Signaling Architecture) was launched on SpaceX mission CRS-5 on 10 January 2015. Dry, sterilized Arabidopsis seeds were planted aseptically on the surface of 10-cm2 solid media plates and remained dormant until removed from cold stowage and exposed to light at the initiation of the experiment on the ISS (International Space Station). The plates were grown in the Vegetable Production System (VPS/Veggie) hardware on the Columbus Module of the ISS with the overhead LED lighting of the VPS. 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. Upon return to Earth, the harvested material was used to compare the transcriptomes of each genotype and each age using RNAseq technology. 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. 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.
Sample: Ground Control, 4 days old, WS Rep1
SAMN06465177 • SRS2020265 • All experiments • All runs
Library:
Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction 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.
Experiment attributes:
GEO Accession: GSM2509423
Links:
Runs: 20 runs, 20.3M spots, 2.9G bases, 1.2Gb
Run# of Spots# of BasesSizePublished
SRR52856271,474,815213.3M79.9Mb2018-02-25
SRR5285628960,720138.3M65.2Mb2018-02-25
SRR52856291,006,425144.8M65.6Mb2018-02-25
SRR5285630996,458143.4M65.3Mb2018-02-25
SRR5285631979,811141.1M64.4Mb2018-02-25
SRR5285632913,059131.4M62Mb2018-02-25
SRR5285633984,851141.8M63.8Mb2018-02-25
SRR5285634954,936137.4M63Mb2018-02-25
SRR5285635848,988122.6M56Mb2018-02-25
SRR5285636842,767121.5M55.6Mb2018-02-25
SRR5285637843,028121.7M54.8Mb2018-02-25
SRR52856381,463,755211.6M79.4Mb2018-02-25
SRR5285639867,501125.2M55.3Mb2018-02-25
SRR52856401,523,465220.3M81.4Mb2018-02-25
SRR52856411,479,356213.9M78.8Mb2018-02-25
There are 5 omitted runs. See all runs in Run Selector.

ID:
3750882

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