GSM1212963: Phytophthora infestans mycelium small RNA rep1; Phytophthora infestans; RNA-Seq
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'.
GEO
GSM1212963