|
| Status |
Public on Jun 22, 2011 |
| Title |
M.oryzae-S-24h |
| Sample type |
SRA |
| |
|
| Source name |
Leaves
|
| Organism |
Oryza sativa |
| Characteristics |
cultivar: Nipponbare
Stage: Biotic stress
tissue: Leaves
|
| Treatment protocol |
For biotic stress treatment; 3 weeks old rice plants were challenged with Magnaporthe oryzae (isolate KJ201) and leaves were collected post inoculation at 6, 12, 24 and 96hrs for total RNA isolation (both wild type Nipponbare and transgenic rice expressing Pi9 blast resistance gene). In case of insect infestation libraries; 2 months old rice plants were challenged with beet army worm and water weevil insects. Leaves were collected 24hr post infestation for total RNA isolation. For abiotic stress libraries, for cold treatment plants were stressed at 40C 24 h; for drought stress, plants were stressed without water for 5 d; for salt stress, plants were stressed by immersing in 250 mM NaCl solution for 24 h. All rice tissues were harvested when the plants were in darkness (4 hours after end of the ‘day’) to minimize to concentration of photosynthetic genes.
|
| Growth protocol |
Plants were grown in Conviron growth chambers at 26°C (day) and 20°C (night) (80% humidity, 12-hr-light/dark cycle; 700 µmol photons m-2 sec-1).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA for library construction was extracted using TriZol
Libraries were prepared according to Illumina's instructions accompanying the sample preparation kit (Catalog No. FC-102-1007). Isolate mRNA and synthesize first strand cDNA followed by second strand cDNA. Restriction digestion of the double stranded cDNA was done using DpnII enzyme. GEX DpnII Adapter 1 was ligated at the site of DpnII cleavage. Sixteen base pair tag was created by restriction digestion with MmeI. GEX adapter 2 was ligated at the site of MmeI cleavage. The GEX adapter 2 contains sequences complementary to the oligos attached to the flow cell surface. The adapter-ligated cDNA construct was selectively enriched using 15 cycles PCR. The amplified cDNA construct was purified and the purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
Leaves collected from 3 weeks old wild type Nipponbare plants 24 hours after rice blast (KJ201) inoculation - Sequencing technical replication -1
Library strategy: Illumina's Sequencing by Synthesis
|
| Data processing |
Adapter sequences were removed using a Perl script, generating MmeI-derived tags initiated by a DpnII site (GATC). After this, the non-redundant sequences were generated with abundance for each distinct sequence. These were mapped to the Oryza sativa genome (v6.1 from MSU).
Alignment: Briefly, the "virtual" signatures were derived from the rice genome by extracting all occurrences of GATC plus the 16 nt sequence at the 3' terminus. These signatures were used for matching analysis with the experimental SBS signatures obtained from Illumina genome analyzer.
All the virtual genomic signatures derived from the rice genome were assigned a "class" based on the position of the signature relative to annotated genes. Signatures that did not match to the genome corresponded to the "Class 0" signatures and those that matched the genome corresponded to Classes 1 to 7. The bioinformatics pipeline for the SBS data analysis was performed similar to MPSS data analysis with few modifications (Meyers et al., 2004; Genome Research 14: 1641-1653; Nobuta et al. 2007; Nature Biotechnology 25(4) 473-477). For MC24 and MR24, raw files resulting from three separate runs were processed and concatenated to produce a single processed file.
For MC24 and MR24, raw files resulting from three separate runs were processed and concatenated to produce a single processed file.
|
| |
|
| Submission date |
Nov 24, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Blake C. Meyers |
| E-mail(s) |
bmeyers@danforthcenter.org
|
| Phone |
314-587-1422
|
| Organization name |
Donald Danforth Plant Science Center
|
| Lab |
Meyers lab
|
| Street address |
975 N Warson Road
|
| City |
St. Louis |
| State/province |
MO |
| ZIP/Postal code |
63132 |
| Country |
USA |
| |
|
| Platform ID |
GPL9147 |
| Series (1) |
| GSE25596 |
Deep Transcriptional Profiling of Rice Using Illumina's Sequencing By Synthesis Technology |
|
| Relations |
| SRA |
SRX032227 |
| BioSample |
SAMN00138859 |
