Novaseq of Globodera rostochiensis Re192_Karang_Tengah_Central_Java_2017
Per sample at least 15 cyst were added to 150l MQ water supplemented with 2 iron beads ( = 3mm) in a 1.5ml tube, and samples were bead beaten for 80 s at 30 hz. Homogenized cyst material (not the beads) were transferred to a new 2ml tube pre-filled with 150l MQ 300l 2X nematode lysis buffer (Holterman et al. 2006) was added, and tubes were incubated in a Thermomixer (Eppendorf, Hamburg, Germany) for 1-2 hrs at 60C at 650rpm. Once the sample was cooled back to room temperature, 150 l 5M NaCl was added to the lysate. Subsequently, an equal volume of Phenol: Chloroform: Isoamyl Alcohol (25:24:1) pH8.0 (4C) was added and the resulting suspension was gently mixed, followed by phase separation by centrifugation (2,500 g for 10 min.).  The upper aqueous layer was transferred to new tube, and an equal volume of Chloroform: Isoamyl Alcohol (24:1) (4C ) was added. After gentle mixing, the DNA was spun down by centrifugation (15,000 g for 5 min.). Thereafter, the supernatant was carefully removed. The DNA pellet was washed with 300 l of 70% ethanol and spun down again for 1 min. at 15.000g. The supernatant was removed, and the pellet was dried for 1 min. at 60C. The final, gently dried pellet was resuspended in 50l 10mM Tris-HCL pH8.0