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Sample GSM2493794

Query DataSets for GSM2493794

Status

Public on Jun 14, 2017

Title

Dark grown, Spaceflight, PhyD Rep3

Sample type

SRA

Source name

solid 0.5x MS media plates; grown in spaceflight, 11 day old plant harvested into KFT containing RNAlater fixative

Organism

Arabidopsis thaliana

Characteristics

cultivar: PhyD
tissue: Roots
organism part: root tip - the last 1mm of the root for the dark grown plants
age: 11 days old plants
protocol: dark grown
biosource type: RNAlater fixed_sample

Treatment protocol

The dormant plates were activated on station by removing the Black-Out cloth wrapping 12 days after launch. The plates were then placed on a fabric that was mounted in the US Laboratory module on the wall adjoining the MELFI freezer and secured using Velcro. The plants were allowed to grow on orbit for 11 days; some in the ambient light of ISS and some in the dark. The dark-grown plates were first activated by exposing the seeds to light for 4 hours, and then re-wrapped in Black-Out cloth for the duration of the growth period. A corresponding set of seedlings were grown as ground control in KSC. At 11 days, seedlings were photographed, harvested into KFT containing RNAlater solutions and returned for post-flight analysis. All plates were harvested into KFTs with their counterpart (e.g. Light 1 was harvested with Dark 1). 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, the -32°C freezer. Following SpaceX-3 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 on May 22, 2014. The PIs retrieved the plant samples from the KFTs on May 29, 2014 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 among genotypes within a treatment (spaceflight or unit gravity) and between treatments (spaceflight versus ground control) for each genotype.

Growth protocol

Three seed lines Wild-Type Wassilewskija (Ws), Columbia-0 (Col-0) and Col-0 PhyD (phyD) Mutants were tested for viability, sterility and ability to maintain dormancy before the launch. Tested batches of seeds were planted on phytagel plates as one genotype per plate for gene expression analysis in replicates of three. One set was planted for the flight and one for ground control. The plates were wrapped such that every surface of the plate was covered by two layers of Duvetyn Black-Out cloth (Seattle Fabrics) (Sng et al, 2014). The plates were stored 4° C until launch, and was then launched in a cold-stow bag to maintain the plates at 4° C until integration and activation on the ISS.

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, root tip and then the rest of the root. The root tip was defined as the last 2mm of the root for the light-grown plants and the last 1mm for the dark-grown plants. All the sections dissected were saved in RNA later and stored back to -80°C freezer. Three independent roots were used as three biological replicates for the transcriptomic analysis. The root apex was dissected as 2mm root tip section for light grown samples and 1mm section for the dark grown samples. Dissected root tips were carefully transferred into pre-labeled micro-centrifuge tubes, flash frozen in liquid N. Total RNA was extracted from these frozen samples using Picopure RNA isolation kit (Arcturus®, Life technologies). RNA concentration was determined on a Qubit (Thermo Fisher/Life Technologies, Inc) and sample quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). Five ng RNA from each sample was used for low input array and RNA sequencing.
RNASeq Libraries Preparation using SMART-Seq V4 ultra low input RNA kit for sequencing combined with Illumina Nextera DNA Sample Preparation Kit Five 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 9 PCR cycles. Then Illumina sequencing libraries were generated with 120 pg of cDNA using Illumina Nextera DNA Sample Preparation Kit (Cat#: FC-131-1024) according to manufacturer’s instructions. Briefly, 120 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). Finallly, the libraries were pooled equal molar concentration and sequenced by Illumina 2X75 NextSeq 500.
NextSeq500 sequencing: RNA seq 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 three flowcells, 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

DK FLT PhyD Rep3
8. DK FLT Col-0 to DK FLT PhyD
14. DK FLT PhyD to DK GC PhyD
20. DK FLT PhyD to LT FLT PhyD

Data processing

The paired end reads were mapped to the A. thaliana var. Columbia reference genome (TAIR10) using TopHat (Trapnell et al., 2009).
Multi-step process of transcriptome assembly and differential expression analysis was done using the Cufflinks tool (Trapnell et al., 2010; Trapnell et al., 2012). Reads that map to each transcript were counted and normalized based on fragment length and total reads. Normalized counts were expressed in terms of FPKM values (fragments per kilobase of transcript per million mapped fragments). FPKM is directly proportional to abundance of the transcript. The expression data was generated at a False Discovery Rate = 0.01 (FDR=0.01). At this FDR, a total of 33,234 genes were tested.
Genome_build: TAIR10
Supplementary_files_format_and_content: FPKM, fold change

Submission date

Feb 16, 2017

Last update date

May 15, 2019

Contact name

Robert J. Ferl

E-mail(s)

ferllabuf@gmail.com

Phone

352-273-8030

Organization name

University of Florida