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Status |
Public on May 18, 2017 |
Title |
UV_III_7 |
Sample type |
SRA |
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Source name |
UV_zone 3
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Organism |
Zea mays |
Characteristics |
cultivar: B73 inbred exposed to: UV-B radiation tissue: Zone 3 (2-3cm from base of the leaf) replicate: Biological replicate 1
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Treatment protocol |
UV treatments were done using different plastic filters to screen UV-B. Solar UV-B radiation was removed to produce the minus UV-B (Control) treatment using PE filters (100 mm clear PE plastic, Tap Plastics, Mountain View, CA). This PE filter absorbs UV-B without significantly affecting UV-A or visible radiation. To control for differences in wind or humidity under plastic sheeting, CA sheeting was used (100 mm extra clear CA plastic, Tap Plastics); the CA sheeting transmits most radiation from sunlight and is designated as the full UV treatment. For this experiment, 1 x 2 m of each plastic was draped over 0.5 x 1.4-m wooden frames that were erected outdoors; the excess plastic was stapled to the sides of the frames to reduce shading. The North and South sides were left open to allow air to circulate. However, 50-cm-long curtains with the same plastic were made on the east and west sides to avoid early morning and late afternoon UV exposure. The frames were maintained about 30 cm above the plant canopy during the experiments. Measurements of incoming UV-B radiation were recorded at noon using a UV-B/UV-A radiometer (UV203 A+B radiometer, Macam Photometrics, Ltd, Livingston, UK). The average UV-A radiation was 8 W/m2 in both groups and the average UV-B radiation was 0.35 W/m2 in the control group and 1.5 W/m2 in the UV-B exposed group. The experiment was repeated three times during the summer of 2013.
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Growth protocol |
Maize (Zea Mays) plants from the B73 inbred were used in filed experiments during the summer 2013 in Rosario, Argentina (32º latitude),
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated from the first three10-mm leaf segments from the basal 30 mm of leaf 4 three days after leaf emergement, during steady-state growth, from each treatment. The TRIzol reagent (Invitrogen) was used as described in the manufacturer’s protocol. Then, 0.5 to 1.0 mg of total RNA was incubated with RNase-free DNase I (1 unit/mL) following the protocol provided by the manufacturer to remove possible genomic DNA. Prior to library preparation the RNA quality and integrity was assessed by using a gel cartridge on a QIAxcel platform (Qiagen, Hilden, Germany). Library preparation was done using the TruSeq® Stranded mRNA sample preparation 96 rcx kit (Illumina™) following the low sample protocol according to Illumina™ guidelines. Briefly, approximately 2.5 µg of total RNA was diluted and purified using RNA purification beads targeting the poly-A tail of the mRNA and subsequently was fragmented by means of the enzymes provided in the kit. After the cDNA synthesis adenylation of 3’ ends and ligation of the adaptors were employed. Adaptors were ligated in 12-plex formations, allowing the pooling of 12 samples after the PCR enrichment of the library. Subsequently, the library was quantified using PicoGreen® dye (Life Technologies™) as described in the manufacturer’s protocol. In order to accurately quantify the concentration in nM of our pools, the Kapa SYBR® FAST universal qPCR kit (Kapa Biosystems™) for Illumina™ sequencing was used to quantify the number of the amplifiable molecules in the pools and the Bioanalyzer® (Agilent Technologies™) to determine the average fragment size of our pools. These measurements allowed optimizing the flow cell clustering and proceed with the Run. The illumina HiSeq 1500 was used in the high throughput mode together with the TruSeq® SBS Kit to sequence the samples in a 25 cycle pair-end run. Following the analysis of these runs, rapid run was performed on the samples having yielded below 5 million reads. The rapid runs were performed on the same pools as in the first sequencing round. Data from these rapid runs were added to the high throughput data. Groups of 12 samples were pooled at equal concentrations to create eight pools for each of the lanes of the flow cell
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 1500 |
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Data processing |
Trimming of Illumina adapters (CLC Genomics Workbench v.6 ) Quality control for individual samples (CLC Genomics Workbench v.6 ) Mapping against the reference genome (CLC Genomics Workbench v.6 ) Calculation of expression values using unique matches (CLC Genomics Workbench v.6) Normalisation by scaling 10.000.000 reads per sample Genome_build: Zea mays (cv B73) sequence (RefGen V3; http://www.maizegdb.org)
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Submission date |
Mar 09, 2017 |
Last update date |
May 15, 2019 |
Contact name |
Gerrit T.S. Beemster |
E-mail(s) |
gerrit.beemster@uantwerpen.be
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Phone |
+32 3 265 34 17
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Organization name |
University of Antwerp
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Department |
Biology
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Lab |
Molecular Plant Physiology and Biotechnology
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Street address |
Groenenborgerlaan 171
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City |
Antwerpen |
State/province |
Antwerpen |
ZIP/Postal code |
2020 |
Country |
Belgium |
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Platform ID |
GPL22940 |
Series (1) |
GSE95858 |
Gene expression analysis of the effect of UV-B radiation in the growth zone of the maize leaf |
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Relations |
BioSample |
SAMN06554718 |
SRA |
SRX2627993 |
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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