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Sample GSM2516761

Query DataSets for GSM2516761

Status

Public on Jun 07, 2019

Title

Ground Control, 8 days old, Sku6 Rep3

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: Sku6
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, 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 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_Sku6_to_GC_8d_Sku6
GC_4d_Sku6_to_GC_8d_Sku6
GC_8d_Sku6_to_GC_8d_Col-0

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 Col-0 TAIR10 genome 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 01, 2017

Last update date

Jun 07, 2019

Contact name

Robert J. Ferl

E-mail(s)

ferllabuf@gmail.com

Phone

352-273-8030

Organization name

University of Florida