bacterial symbionts of the pinewood nematode
DNA were isolated from enriched bacteria isolated from the PWN strain ZJSS. At first, an enrichment of bacterial symbionts of the nematodes was performed using Nycodenz density gradient centrifugation. Briefly, nematodes were gently homogenized in DDW. Nycodenz solution (Axis shield, Oslo, Norway) with a density of 0.387 g/ml was added to the homogenate, and then centrifuged with 12,000 rpm/min for 15 minutes at 4oC. The cell suspension was withdrawn and washed several time with DDW to remove Nycodenz. Then, the bacterial cells merged with enrichments of several times were suspended and used for DNA extraction. Sodium dodecyl sulfate (SDS) was added (to a final concentration 0.5%) for lysis the remains of nematode cells at 0oC for 1 hour. After centrifugation, a lysis solution (200 mmol/L Tris.HCl, pH8.0, 200mmol/L NaCl, 100 mmol/L EDTA, 2% SDS and 500µg/ mL protease K) was added to the bacterial cells suspension and at 65oC for 3 hours. Phenol-chloroform method was used to extract the bacterial genomic DNA. DNA concentration and purity was assessed by agarose gel electrophoresis and spectrophotometric analysis. DNA extracted from three times was mixed together and used for the library construction.  About a 20-µg genomic DNA was used for a shotgun DNA library construction. DNA library preparation followed the manufacturer’s instruction (Illumina). The same workflow as described by Qin (Qin et al, 2010) was performed, with both ends sequencing (clone insert size about 200bp) by the Illumina Genome Analyzer at the Beijing Genomics Institute, Shenzhen, China.