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| Status |
Public on Jul 20, 2020 |
| Title |
B3-CK |
| Sample type |
SRA |
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| Source name |
Grapevine berry at 10 DAF
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| Organism |
Vitis vinifera |
| Characteristics |
tissue: Grapevine berry
age: at 10 DAF
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| Treatment protocol |
In order to investigate a demonstrative biological selection of transcripts at green-berry stage, RNA for Illumina sequencing was filtered from tissues comprised of three biological replicated for this stage.
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| Growth protocol |
Three-year-old ‘Fujiminori’ grape trees grown in Nanjing, China (N32°02′12.77′′, E118°37′33.25′′) were chosen as the experimental material. The plant materials were grown under common field conditions at the Jiangsu Vocational College of Agriculture and Forestry grape farm, Jurong, China.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA of the berry samples at GBS was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the processed RNA was checked for purity and integrity using Nanodrop-2000spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).
A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Raw data(raw reads) of fastq format were firstly processed through in-house peri scripts. In this step, clean data(clean data) were obtained by removing reads cotaining adapter.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and iscurrently the most commonly used method for estimating gene expression levels.
Genome_build: https://genomes.cribi.unipd.it/grape/search_form.php
Supplementary_files_format_and_content: FPKM
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| Submission date |
Jun 24, 2020 |
| Last update date |
Jul 20, 2020 |
| Contact name |
Wenran Wang |
| E-mail(s) |
[email protected]
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| Organization name |
Nanjing Agricultural University
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| Street address |
Weigang
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| City |
Nanjing |
| ZIP/Postal code |
210095 |
| Country |
China |
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| Platform ID |
GPL18580 |
| Series (1) |
| GSE153169 |
Genome-wide identification and characterization of Gibberellin Metabolic and Signal Transduction (GA MST) pathway mediating seed and berry development (SBD) in Grape (Vitis vinifera L.) |
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| Relations |
| BioSample |
SAMN15357956 |
| SRA |
SRX8609960 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4635174_B3_CK.txt.gz |
1.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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