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| Status |
Public on Jul 28, 2013 |
| Title |
Mutant-Mar-S-2 |
| Sample type |
SRA |
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| Source name |
mutant_anthers
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| Organism |
Gossypium hirsutum |
| Characteristics |
background: Dong A
genotype/variation: Genetic male sterility (GMS) mutant
developmental stage: tetrad stage
tissue: anthers
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| Treatment protocol |
According to this sampling criterion and combined with microscopic examination, developing anthers at these three different growth stages were collected during early mornings.
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| Growth protocol |
Upland cotton (G. hirsutum) ‘Dong A’ (WT) plants and the GMS mutant in the ‘Dong A’ background were grown under regular field conditions at the experimental farm of the Cotton Research Institute in China Agricultural Academy of Science.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from anthers using the pBiozol Total RNA Extraction Reagent (BioFlux), in accordance with the manufacturer’s instructions. The RNA was then precipitated with ethanol, dissolved in diethypyrocarbonate (DEPC) water and stored at −80°C. All RNA samples were examined for protein contamination (A260/A280 ratios) and reagent contamination (A260/A230 ratios) using a Nanodrop ND 1000 spectrophotometer (NanoDrop, Wilmington, DE).
The samples from the WT and GMS mutant anthers were quantified and equalized so that equivalent amounts of RNA were analyzed. The extracted total RNA was resolved on denatured 15% polyacrylamide gels. Gel fragments with a size range of 18–30 nt were excised, and the small RNA fragments were eluted overnight with 0.5 M NaCl at 4°C, and precipitated with ethanol. These 18–30 nt small RNAs were given 5’ and 3’ RNA adapters that were ligated with T4 RNA ligase. The adapter-ligated small RNAs were subsequently transcribed into cDNA by Super-Script II Reverse Transcriptase (Invitrogen) and amplified with the polymerase chain reaction, using primers that were annealed to the ends of the adapters. The amplified cDNA products were purified and recovered. Finally, Solexa sequencing technology was employed to sequence the small RNA samples (BGI, Shenzhen, China).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
Small RNA
Sample 5
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| Data processing |
Raw sequence reads were produced using an Illumina 1G Genome Analyzer at BGI (Shenzhen, China) and processed into clean full-length reads through the BGI small RNA pipelines.
During this procedure, all low quality reads were removed, such as reads with 3’ and 5’ adapter contaminants, those without insert tags, and those with poly A sequences.
The remaining high-quality sequences were trimmed of their adapter sequences, and those larger than 30 nt or smaller than 18 nt were discarded. All high-quality sequences, even those with only a single unique read, were considered significant and used for further analysis.
The small RNAs were aligned to miRNA precursors/mature miRNAs in the miRBase (http://www.mirbase.org/index.shtml, release 15.0). The following criteria were used to determine the sequence counts of miRNA families in the different tissue samples: (1) if there was cotton miRNA information in the miRBase, the small RNAs were aligned to the corresponding cotton miRNA precursor/mature miRNA; and (2) if there was no cotton miRNA information in the miRBase, the small RNAs were aligned to the miRNA precursors/mature miRNAs of all plants in the database.
To identify potentially novel miRNAs among the six small RNA libraries, cotton transcript assemblies (ftp://occams.dfci.harvard.edu/pub/bio/tgi/data/Gossypium) from the Dana Farber Cancer Institute were chosen to map unique small RNA sequences. The characteristic hairpin structure of miRNA precursors was used to predict possible novel miRNAs. The miRNA prediction software, mireap, was also used to predict novel miRNA based on the secondary structure, the Dicer cleavage site, and the minimum free energy (http://sourceforge.net/projects/mireap/).
Genome_build: CGI.release_11.zip at ftp://occams.dfci.harvard.edu/pub/bio/tgi/data/Gossypium
Supplementary_files_format_and_content: Text files include lengths and counts of each distinct reads.
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| Submission date |
Jan 22, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Ming ming Wei |
| E-mail(s) |
[email protected]
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| Phone |
86-372-2525365
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| Organization name |
Cotton Research Institute, Chinese Academy of Agriculture Sciences (CAAS)
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| Street address |
Huang he road 38
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| City |
Anyang |
| ZIP/Postal code |
455000 |
| Country |
China |
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| Platform ID |
GPL9362 |
| Series (2) |
| GSE43531 |
Comparative expression profiling of miRNA during anther development in genetic male sterile and wild type cotton [small RNA] |
| GSE43532 |
The role of microRNA-regulating energy metabolism and pollen wall development in male sterility in cotton |
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| Relations |
| SRA |
SRX218729 |
| BioSample |
SAMN01893765 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1067616_Small_RNA_sequencing_processed_profile-Mar-S-2.txt.gz |
65.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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