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| Status |
Public on Jul 28, 2013 |
| Title |
Degradome library |
| Sample type |
SRA |
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| Source name |
Upland cotton_anthers pooled
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| Organism |
Gossypium hirsutum |
| Characteristics |
line background: Dong A
genotype/variation: mixture of WT and GMS mutant
tissue: Anthers of the WT and GMS mutant representing three stages of development
develpmental stage: mixed (meiosis, tetrad,and uninucleate microspore stage)
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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 |
In this study, in order to dissect miRNA-guided gene regulation in the WT and GMS mutant anthers, a degradome library suitable for miRNA target identification was constructed as described previously.
Briefly, total RNAs, which were extracted from the WT and GMS mutant anthers representing three stages of development, respectively, were mixed at an equal molar ratio as one sample. Approximately 200 μg of the mixed total RNA was used for polyadenylation using the Oligotex mRNA mini kit (Qiagen). Using T4 RNA ligase (Takara), a 5’ RNA adapter was added to the cleavage products, which possessed a free 5’-monophosphate at their 3’ termini. The ligated products were then purified using Oligotex mRNA mini kit (Qiagen) for reverse transcription to generate the first strand of cDNA using an oligo dT primer via SuperScript II RT (Invitrogen). After the cDNA library was amplified for 6 cycles (94°C for 30 s, 60°C for 20 s, and 72°C for 3 min) using Phusion Taq (NEB), the PCR products were digested with restriction enzyme Mme I (NEB). A double-stranded DNA adapter was then ligated to the digested products using T4 DNA ligase (NEB). The ligated products were selected based on size by running 10% polyacrylamide gel and purified for the final PCR amplification (94°C for 30 s, 60°C for 20 s, and 72°C for 20 s) for 20 cycles. The PCR products were gel purified and used for high-throughput sequencing using Illumina HiSeq 2000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Low quality sequences and adapters were removed before sequence analysis.
Unique sequence signatures were aligned to the database of cotton transcript assemblies in Cotton Gene Index (Release 11.0, http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=cotton).
The CleaveLand was used to detect potentially cleaved targets based on degradome sequences. The 20 and 21 nt distinct reads were subjected to the CleaveLand pipeline for small RNA targets identification as previously described [Addo-Quaye C, Miller W, Axtell MJ: CleaveLand: a pipeline for using degradome data to find cleaved small RNA targets. Bioinformatics 2009, 25: 130-131].
the 20 and 21 nt distinct reads were first normalized to give 'reads per million' (RPM). Subsequently, the degradome reads were mapped to the cotton annotated cDNA (DFCI-Cotton Gene Index, release 11.0) and the cDNA hit number of each degradome read was recorded.
All alignments with scores not exceeding 4 and having the 5’ end of the degradome sequence coincident with the tenth and eleventh nucleotides of complementarity to the small RNA were retained.
Genome_build: DFCI-Cotton Gene Index Release 11.0 at http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=cotton
Supplementary_files_format_and_content: Text files include lengths and counts of each distinct reads.
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| Submission date |
Jan 09, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Ming ming Wei |
| E-mail(s) |
lywmm@yahoo.cn
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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 |
GPL16485 |
| Series (2) |
| GSE43389 |
Comparative expression profiling of miRNA during anther development in genetic male sterile and wild type cotton [degradome] |
| GSE43532 |
The role of microRNA-regulating energy metabolism and pollen wall development in male sterility in cotton |
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| Relations |
| SRA |
SRX216096 |
| BioSample |
SAMN01884269 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1061853_Degradome_library-processed_data.txt.gz |
67.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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