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| Status |
Public on Oct 27, 2018 |
| Title |
ROOT ZONE I (0.5mm) REP 2 [RNA-seq] |
| Sample type |
SRA |
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| Source name |
solid media plates; 24 hr light grown
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| Organism |
Arabidopsis thaliana |
| Characteristics |
cultivar: Col-0
genotype/variation: WT
age: 8 days old plants, light grown
tissue: Roots
tissue subtype: 0.5mm from the tip including the root cap
biosource type: RNAlater fixed_sample
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| Treatment protocol |
Plants were grown for 8 days
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| Growth protocol |
Arabidopsis thaliana var. Columbia (COL-0) seedlings were grown on sterile solid media plates containing 0.5 % phytagel. The plates were vertically placed in growth chambers with continuous light (80-100 µmol m -2) at a constant temperature of 19° C. Two Arabidopsis thaliana Col-0 cell culture lines were used: Wild Type (WT) and ARG1 KO SALK_024542C line (ARG1 T-DNA insertion knockout), all cell cultures derived from hypocotyls. Tissue culture cells were plated on 60mm round, nutrient agar Petri plates cultured in a horizontal position until turnover and installation into the BRIC hardware. All of the sub-culturing on the plates was conducted in the laboratory of the PIs and the University of Florida. Plates were planted in a laminar flow hood and stored in sterile Biotransfer Boxes (BT). Plates with cells were kept in the dark and at ambient temperature. Preparation of specialized plates and biology Petri plates. Because of their configuration within the BRIC flight hardware, sterility was maintained on all surfaces (interior and exterior) of the Petri plates. The media in the plates for BRIC had to be 5mm deep or less to accommodate the biology and the PDFU cover hardware. For a 60mm dish, this calculates out to 6.7 mLs. After pouring, the plates are allowed to solidify, and then transferred to a sterile Biotransporter (to maintain sterility of external surface) until planting. Preparation of cell culture plates. The liquid media from a sterile cell suspension was decanted, and the cells were washed once with fresh liquid media, and then decanted again. A sterile scoop was used to place about 1 gram of cells in the surface on the plate and disperse evenly across the surface. The cover was replaced, but not taped, and transferred to a sterile BT. The BT was wrapped in black cloth and stored at room temperature. The same day plates were transported by car to the Kennedy Space Center, NASA for the same night (3:00 AM) Turn Over. The ground control plates were turned over 48 hours later. BRIC Spaceflight Hardware The hardware for this experiment was the NASA BRIC-LED (Biological Research In a Canister – Light Emitting Diode). The LED component of the hardware was disabled and all biology was held in the dark for the duration of the experiment. The BRIC-LED canisters hold six 60 mm diameter Petri plates in small sub-compartments called PDFUs (Petri Dish Fixation Units). The PDFUs allow an externally activated fixation step without manipulating the samples. We were allocated 10 PDFUs: 5 in BRIC A and 5 in BRIC B. The 2 replicate plates of WT cultured cells and the 3 replicate plates of ARG1 KO were distributed among the BRIC A canister. The 2 replicate plates of WT cultured cells were distributed among the BRIC B canister. One of the PDFU slots in each BRIC was used to hold a HOBO™ data logger to record temperature. The HOBO temperature data collection begins immediately after the biology is sealed in the BRICs, 24 hours before launch. An additional set of Petri plates within BRIC hardware, identical to that launched for Spaceflight, was prepared for the Ground Control. The Ground Control was housed in the OES (Orbital Environmental Simulator) chamber in the Spaceflight Life Sciences Laboratory (SLSL) at Kennedy Space Center (KSC). The Ground Control was initiated with a precise 24 hour delay to enable the OES environment to be programmed with the environmental profile taken from telemetry of the Space Shuttle. Nominally, the Ground Control BRIC hardware and samples experienced the same external environment profile as the hardware and samples on the Space Shuttle. Environmental parameters that are controlled on the OES and made identical to flight are cabin temperature, humidity and CO2 concentration.
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| Extracted molecule |
total RNA |
| Extraction protocol |
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.).
Library preparation: Five ng of total RNA was used for cDNA library construction using CloneTech SMART-Seq V3 ultra low input RNA kit for sequencing (Clontech Laboratories, Inc,) according to manufacturer's protocol. Briefly, 1st strand cDNA was primed by the SMART-SMARTer II A 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 10 PCR cycles. Then Illumina sequencing libraries were generated with 120 pg of cDNA using Illumina Nextera DNA Sample Preparation Kit (Cat#: FC-131-1024) per manufacturer’s instructions. Briefly, 120 pg of cDNA was fragmented by tagmentation 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 by equal molar concentration and sequenced by Illumina 2X150 NextSeq 500 (Illumina Inc., CA, U.S.).
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 a single flowcell, using a 2x150 cycles (paired-end) configuration. A typical sequencing run in the NextSeq500 produced 750-800 million paired-end reads with a Q30>=85%. For RNA seq, around 40 million reads/sample provided sufficient depth for transcriptome analysis.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Z1 Rep 2
C2-2_S2
processed data file:
RNAseq_Genediff_pilot
RNAseq_Isoformsdiff_pilot
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| Data processing |
The paired end reads were mapped to the A. thaliana var. Columbia reference genome (TAIR10) using TopHat. Multi-step process of transcriptome assembly and differential expression analysis was done using the Cufflinks tool. 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.
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.
Genome_build: TAIR10
Supplementary_files_format_and_content: differential gene expression
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| Submission date |
Jun 11, 2018 |
| Last update date |
Oct 27, 2018 |
| 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 |
GPL19580 |
| Series (2) |
| GSE115554 |
Comparative gene expression analysis in the Arabidopsis thaliana root apex using RNA-seq and microarray transcriptome profiles [RNA-seq] |
| GSE115555 |
Comparing RNA-Seq and microarray gene expression data in two zones of the Arabidopsis root apex relevant to spaceflight |
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| Relations |
| BioSample |
SAMN09389430 |
| SRA |
SRX4190902 |
| 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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