Pernospora effusa race 8 genomic sequencing reads
P. effusa spores were harvested from non-wilting leaves that contained sporulating from spinach plants and brought into suspension in tap-water. Soil and other large contaminants were removed by filtering the spore suspension over a 50m nylon mesh filter followed by a filtering over a 11 m nylon mesh filter using the Merck All-Glass Filter Holder (47mm) and a vacuum pump. Remaining spores were resuspended in 100 mL of autoclaved tap water and filtered again. This washing step was repeated 10 times. P. effusa spores were freeze-dried and lysed using glass beads in a tissue lyser (2 x 30 seconds on 30 Hz). The lysed spores were incubated in extraction buffer (3% CTAB, 2% PVP, 1.25 M NaCl, 200 mMTris. HCl, pH 8.5, 25 mM EDTA, pH 8.0) for 30 minutes at 65 C. Then RNase was added, and the sample was incubated for 30 minutes at 37 C, and then 0.5% 2-Mercaptoethanol was added, and the sample was incubated again for 15 minutes at 37 C. Afterwards, the DNA was isolate by a phenol/chloroform/IAA extraction, a chloroform/IAA wash, RNase treatment, another phenol/chloroform/IAA extraction, a chloroform wash, and isopropanol precipitation. Isolated DNA sampel was quantified with the Qubit 2.0 fluorometer using the Qubit dsDNA HS (High Sensitivity) assay kit. The sequencing library was constructed with the Illumina TruSeq DNA PCR-Free kit. Fragment-size distribution in the library was determined before and after the library preparation using the Agilent Bioanalyzer 2100 with HS-DNA chip (Agilent Technologies). The library was sequenced on an Illumina Nextseq machine in high output mode with a 550 bp genomic insert paired-end 150 bp reads.