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| Status |
Public on Oct 31, 2013 |
| Title |
Ve2 |
| Sample type |
SRA |
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| Source name |
wild type
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| Organism |
Oryza sativa Japonica Group |
| Characteristics |
cultivar: Nipponbare x HEG4 RI line
tissue: pollen minus sperm cells
genotype: wild type
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| Growth protocol |
Egg cells were isolated using flowers harvested at anthesis from rice plants (Oryza sativa L. ssp. japonica, cultivar “Kitaake”) grown in greenhouses under ambient light during the summer. Sperm and pollen vegetative cells were isolated from near anthesis anthers of vigorous visibly disease-free field grown rice at Dale Bumpers National Rice Research Center in Stuttgart, AR, USA.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For egg cell processing, ovaries were mechanically dissected from emasculated flowers under a dissecting scope, moved to fresh 0.3M mannitol (Sigma) prepared using DEPC-treated water (Sigma) and bisected using a double-edged Merkur (Solingen, Germany) razor blades in 3μL drops of 0.3M mannitol. Using an insect pin, the base of the ovary was gently pushed towards the cut surface until the egg cell became visible. Egg cell protoplasts were then immediately transferred to 1.5mL Eppendorf tubes in liquid nitrogen and stored at -80°C. For sperm cell and vegetative cell cytoplasm sample processing, a differential centrifugation isolation method was used to separate the sperm cells and vegetative cells from whole anthers (Gou et al. 1999, Russell et al. 2012). Briefly, collected anthers were gently ground in cold 45% (w/v) sucrose solution to release mature pollen grains, filtered through 100 μm nylon mesh, resuspended in a 10× volume of 15% (w/v) sucrose solution for 20 min to release the sperm cells, filtered through a 30μm nylon mesh and layered on a Percoll density gradient, centrifuged and recovered as described previously (Russell et al. 2012). The precipitate consisting of vegetative cell materials was treated as a sperm depleted sample and collected in concert with each sperm cell isolation. Therefore, the sperm and vegetative samples represent paired samples, but these were treated as independent when analyzed in conjunction with the egg cell samples. Samples collected were immediately transferred and stored in -80°C until use.
RNA from the sperm and vegetative cells was extracted using the QIAGEN RNeasy Plant Mini Kit (QIAGEN) following manufacturer’s instructions including the optional on-column DNAse treatment. For the egg cell samples, RNA was extracted using the RNAqueous-Micro Kit (Applied Biosystems). The protocol was modified to add an on-column DNAse treatment (QIAGEN) after the first wash step. RNA quality for all samples was assessed and concentrations measured using the Agilent Bioanalyzer (Agilent), and samples had an RNA Integrity Number (RIN) ranging from 7.7 – 9. cDNA synthesis was performed using the NuGEN RNA-seq system V2 (NuGEN) with an input of 1.5 – 10 ng of RNA. cDNA was quantified on the ND 1000 Nanodrop Spectrophotometer (Thermo Fisher Scientific, Inc.) and then sheared on the Covaris S220 system (Covaris) using manufacturer’s recommended settings for 200 bp fragments. 170 ng of sheared cDNA was purified using the MinElute Reaction Cleanup Kit (QIAGEN), and libraries were prepared using the NuGEN Ovation Ultralow DR Multiplex Systems 1-8. Samples were multiplexed and run with 6 samples per lane on the Illumina HiSeq 2000 DNA sequencer (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Transcriptional profiling
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| Data processing |
Illumina CASAVA_v1.8.0 software used for basecalling.
The barcoded Fastq files of the reads were mapped to rice RGAP 7.0 genomic reference sequences using Tophat version 2.0.5 with the default settings except that the 'microexon' search option was switched on, and minimum and maximum intron sizes were set at 20 bases and 15000 bases, respectively. The mapped reads in the resulting accepted_hits.bam files were assigned to gene exons of the different isoforms of the RGAP 7.0 gene models GFF3 file, using an in-house Perl script.
For gene analysis only uniquely mapped read counts that were assigned to gene exons were used. Additionally, where more than one isoform is present, the one with the highest number of reads across the three cell types assessed was analyzed further.
To determine differentially expressed genes between the three cell types, the Bioconductor R package edgeR (Robinson et al. 2010) was used. However, the default normalization method, TMM, assumes that most genes are not differentially expressed between conditions, which was clearly not the case with the three cell types, and so the upper quartile method (75th percentile) was used. A glm was fitted to the data with tag-wise dispersion. The following contrasts were created:- EC vs Sp, Sp vs Ve, Ve vs EV, EC vs (Sp + Ve)/2, Sp vs (Ve + EC)/2 and Ve vs (EC + Sp)/2. Likelihood ratio tests were performed to determine differential expression for each contrast.
Genome_build: RGAP rice vs 7.0
Supplementary_files_format_and_content: The comma separated values (csv) file contains count data for reads mapping to exons for alternative transcript isoforms for Oryza sativa rice gene models for LOC_ loci from RGAP version 7.0
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| Submission date |
Sep 11, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Venkatesan Sundaresan |
| Organization name |
University of California Davis
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| Department |
Plant Biology
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| Lab |
Sundaresan
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| Street address |
One Shields Ave
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| City |
Davis |
| State/province |
CA |
| ZIP/Postal code |
95616 |
| Country |
USA |
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| Platform ID |
GPL13834 |
| Series (1) |
| GSE50777 |
Transcriptomes of isolated rice gametes characterized by deep sequencing: Evidence for distinct sex-dependent chromatin and epigenetic states before fertilization |
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| Relations |
| BioSample |
SAMN02354050 |
| SRA |
SRX348619 |
| 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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