Genomic of Populus x canadensis: leaves: Sample GWR_100_475
"Leaf samples were processed for DNA extraction and preparation of sequencing libraries. DNA extraction was performed using the Qiagen DNeasy kit (Qiagen) following the manufacturer's recommendations, except that 4 microliters of proteinase K (20 mg proteinase K in 10 mM Tris-HCl, 20 mM CaCl2, 50% glycerol) was added to the lysis buffer and the mix was incubated at 55 degree Celsius for 30 minutes prior to extraction.Genomic libraries were produced as follows. For each sample, 500 ng of starting DNA was used. Water was added to the DNA to reach a total of 50.4 microliters. The DNA was fragmented by adding 6 microliters of 10X fragmentase buffer, 0.6 microliters of 100X BSA and 3 microliters of DNA fragmentase (New England Biolabs, Ipswich, MA). The mixture was incubated for 45 minutes at 37 C before 5 microliters of 0.5 M EDTA were added to terminate the reaction. The fragmented DNA was purified using AMPure beads (Beckman Coulter Inc, Brea, CA) and a 1:1 ration of beads to sample volumes. End-repair and ""A""-base addition were carried out using the Next DNA Sample Prep kit (New England Biolabs, Ipswich, MA) according to the manufacturer's recommendations, expect that half of the recommended volumes were used for the end-repair reactions. Reactions were purified using AMPure beads and a 1:1 ration of beads to sample volumes, with a final elution volume of 13 microliters. Adaptors for Illumina sequencing were ligated by combining the following: 13 microliters of DNA, 15 microliters of quick ligation reaction buffer, 1 microliters of adaptor mix (50 mm), and 1 microliters of quick T4 DNA ligase and 20 microliters of water for a total of 50 microliters. Adaptors containing 6-bp index (Bioo Scientific, Austin, TX) were used. Size-selection of the resulting fragments were performed using a two-step AMPure purification, as described previously Monson-Miller 2012), with ratios of 1:0.6 and 1: 0.7 volumes of samples to AMPure for the upper and lower cuts respectively and eluted in a final volume of 20 microliters.  The resulting libraries were amplified by PCR by mixing 10 ml of template DNA, 12.5 ml of Phusion High-Fidelity PCR Master Mix (Finnzymes Oy, Espoo, Finland), and 5 ml of 5 mm paired-end primer mix, with the following protocol: 30 sec at 98 degree Celsius followed by 10 cycles of 10 sec at 98 degree Celsius, 30 sec at 65 degree Celsius, and 30 sec at 72 degree Celsius, ending with 5 min at 72 degree Celsius. The PCR products were purified using AMPure beads and a 1:0.8 ratio of beads to sample volumes, with a final elution volume of 15 microliters.Samples were quantified using QuBit 2.0 fluorometer and the High Sensitivity Quantification kit (Invitrogen, Carlsbad, CA) and equimolar amounts of each sample library were pooled prior to sequencing.  Pools of up to 96 samples were sequenced on Illumina HiSeq2000 or HiSeq2500 sequencer (Illumina, San Diego), and 50 single reads were obtained.Monson-Miller, J., Sanchez-Mendez, D.C., Fass, J., Henry, I.M., Tai, T.H. and Comai, L. (2012) Reference genome-independent assessment of mutation density using restriction enzme-phased sequencing. BMC Genomics 13, 72."