T4R1P
Genomic DNA of the wild type and the derivative, transformant strains were extracted via the aqueous / organic extraction method using phenol chloroform. Briefly, the bacteria were grown in C+Y to late logarithmic phase at OD620 ~0.6 and were pelleted at 6000 x g for 10 minutes followed by lysis in 1 mL 1x PBS containing 50 L 10% DOC, 50 l 10% SDS, and 10 L of 10mg/mL Proteinase K (Sigma). The suspension was incubated until clear (approximately 5 minutes) at 37oC, mixed with 500 L phenol : chloroform : isoamyl alcohol (Sigma), transferred to phase-lock tubes (Quantabio), and centrifuged at maximum speed for the separation of aqueous and organic phases. Then 500 L chloroform : isoamyl alcohol was added to and mixed with the aqueous phase and centrifuged. To precipitate DNA, the aqueous phase was transferred to a new Eppendorf tube containing 100% ethanol. Precipitated DNA was washed with chilled 70% ethanol, pelleted, dried, and resuspended in nuclease-free water. Sequence libraries were barcoded using the Nextera kit (Illumina), and run on the Illumina HiSeq2000 platform.