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| Status |
Public on May 10, 2011 |
| Title |
Cotyledon Stage Whole Seed |
| Sample type |
SRA |
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| Source name |
Soybean seeds containing cotyledon-stage embryos
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| Organism |
Glycine max |
| Characteristics |
cultivar: Williams 82
tissue: whole seeds
developmental stage: cotyledon-stage embryo
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| Growth protocol |
Soybean plants (Williams 82) were grown under standard greenhouse conditions (Le et al., 2010). Seed stages were characterized by seed length as follows: globular stage (0.8-1.1 mm), heart stage (2-2.5 mm), cotyledon stage (3-3.9 mm), and early maturation stage (6-7 mm). Dry seeds weighed approximately 200 mg. Seedlings were isolated from seeds imbibed for six days. Vegetative tissues (trifoliate leaves, roots, stems) were isolated from a mature plant 3-4 months old. Floral buds consists of unopened buds with no signs of dehisced anthers.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Collected tissues were quickly frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. Total RNA was isolated from the powder using the Concert Plant RNA Reagent (Invitrogen, Carlsbad, CA) according to the manufacturerâs instructions. Isolated total RNA was treated with RNase-free DNase I (Ambion, Austin, TX) and was subjected to two rounds of poly-A+ RNA selection using oligo-d(T)25 magnetic beads (Dynabeads, Invitrogen, Carlsbad, CA).
Sequencing libraries were prepared using 100 nanograms of twice-selected polyA+ RNA following the Illumina mRNA-Seq Protocol (Part #1004898 Rev. D). Briefly, polyA+ RNA was fragmented and used as template for random-primed cDNA synthesis. Double-stranded cDNAs were treated with T4 and Klenow DNA Polymerases and T4 PNK to generate blunt-end cDNAs. An 'A' base was added to the 3â ends of the phosphorylated blunt-end cDNAs, followed by the ligation of Illumina adapters. Adapter-ligated cDNAs were size selected on an agarose gel for the desired size of ~ 200 bp. Purified cDNAs were amplified by PCR for 15 cycles. Quantification of cDNA libraries was carried out using a Nanodrop ND-3300 Fluoro-spectrophotometer (Thermo Scientific, Waltham, MA). Amplified cDNA size was determined by agarose gel electrophoresis. Single-end 76-bp reads were generated for each library by the UCLA Genome Sequencing Center (http://gsc.ucla.edu/) using an Illumina Genome Analyzer IIx.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Seed lengths ~ 3 â 3.9 mm.
polyA RNA
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| Data processing |
Raw: Original unprocessed and unfiltered data file from the Illumina sequencing pipeline. Each lane of sequencing is attached as an individual compressed file. The sequence data are in the QSEQ format.
Count: Raw sequence reads were initially filtered using the Illumina purity filter parameter. Then, the remaining reads were mapped to a reference containing soybean ribosomal RNA (rRNA) sequences using Bowtie [Langmead et al., Genome Research, (2009)]. The final filtered reads have passed the Illumina purity filter and are devoid of rRNA sequences. These sequences were used to map against the soybean reference genome (Glyma version 1.0.1) [Schmutz et al. Nature (2010)] obtained from the Phytozome website (http://phytozome.net). Only uniquely mapped reads (i.e. reads that map to one position within the genome) were used for subsequent analysis. Predicted gene models with at least one uniquely mapped read were considered transcriptionally active. The total mapped read count for all predicted gene models (66,210 genes) were quantified from all sequencing runs combined.
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| Submission date |
May 09, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Bob Goldberg |
| E-mail(s) |
bobglab@mcdb.ucla.edu
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| Phone |
310-825-3270
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| Organization name |
University of California, Los Angeles
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| Department |
Molecular, Cell and Developmental Biology
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| Street address |
610 Charles E Young Drive East
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
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| Platform ID |
GPL15008 |
| Series (1) |
| GSE29163 |
Genome-Wide Transcript Profiling During Soybean Seed Development and Throughout the Soybean Life Cycle |
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| Relations |
| SRA |
SRX062327 |
| BioSample |
SAMN00265081 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM721727_wm.cot.seed.count.txt.gz |
245.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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