|
| Status |
Public on Oct 21, 2013 |
| Title |
Phytophthora infestans mycelium small RNA rep1 |
| Sample type |
SRA |
| |
|
| Source name |
mycelium_small RNA
|
| Organism |
Phytophthora infestans |
| Characteristics |
strain: T30-4
tissue: mycelium
molecule subtype: small RNA
|
| Growth protocol |
Mycelium was grown from Phytophthora infestans T30-4 maintained on cleared 10% V8 agar medium (100 mL V8 juice; 1 g CaCO3; 30 mg/L β-sitosterol; 15 g agar; 900 mL deionized water) in a 20°C incubator in the dark. Mycelia were grown in V8 broth (V8 agar medium without agar) on a rotating shaker in flasks and collected after 4 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mycelium samples were frozen in liquid nitrogen, ground into a fine powder and homogenized in Trizol (1g:10mL, Life Technologies). After adding 2 mL chloroform per 10 mL Trizol, samples were mixed, incubated at room temperature for 5 minutes and centrifuged at 8400 x g for 10 minutes. Two additional chloroform extractions were done before RNA was precipitated in 0.7 volumes isopropanol for 20 minutes at room temperature and spun for 30 minutes at 8400 x g. Minimally dried pellets were resuspended in 200 μL 0.1X TE, extracted 2 times with phenol:chloroform:isoamyl alcohol (50:49:1), and once with chloroform. Total RNA was precipitated with 5M ammonium acetate and ethanol at -80°C overnight, spun at 12000 x g for 30 minutes at 4°C, resuspended in 100 microliters 0.1X TE and quantitated by Nanodrop (Thermo Scientific).
Small RNA was extracted from total RNA (100 μg) by size fractionation and converted to DNA amplicons by serial adaptor ligation followed by RT-PCR as in (Fahlgren et al., RNA 2009). The 3' adaptor sequence was 5'- CTGTAGGCACCATCAATC-3'.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
Small RNA isolated from total RNA extracted from Phytophthora infestans mycelium
|
| Data processing |
Sequencing reads from small RNA libraries were computationally processed to remove 3’ adaptor sequences, and sequences were consolidated to generate read counts per unique small RNA.
Unique small RNAs were aligned to the Phytophthora infestans genome using Bowtie v0.12.8 with settings allowing for perfect matches only (bowtie –f –v 0 –a –S –p 10).
Genome_build: Phytophthora infestans T30-4, version 1; http://www.ncbi.nlm.nih.gov/assembly/GCA_000142945.1/
Supplementary_files_format_and_content: GFF (version 3) files are provided that contain small RNA mapping data and read abundances (non-normalized) used in our study. GFF formats are specified in the header of each file.
|
| |
|
| Submission date |
Aug 20, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
James C Carrington |
| E-mail(s) |
jcarrington@danforthcenter.org
|
| Phone |
314-587-1202
|
| Organization name |
Donald Danforth Plant Science Center
|
| Lab |
James C. Carrington
|
| Street address |
975 North Warson Road
|
| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63132 |
| Country |
USA |
| |
|
| Platform ID |
GPL17596 |
| Series (2) |
| GSE50030 |
Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs: Phytophthora infestans small RNA |
| GSE50033 |
Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs |
|
| Relations |
| BioSample |
SAMN02319460 |
| SRA |
SRX337389 |
