Drosophila simulans Pennsylania.ECsimPA7_2010 pool of 65 individuals
"We extracted DNA using a lithium chloride precipitation. DNA was prepared for Illumina sequencing by shearing, end- repair and ligation. To do so, 50 mL of DNA was mixed with an additional 50 mL of TE and this DNA was sheared to 500 bp using a Covaris machine. DNA was eluted to 30 mL using a QIAGEN PCR-purification kit (product number 28104). We performed end repair by incubating each sample of DNA with 5 mL T4 DNA ligation buffer, 4 mL of 10 mM dNTPs, 2.5 mL T4 DNA polymerase, 0.5 mL Klenow large fragment, 2.5 mL T4 PNK, and 5.5 mL nuclease free water for 30 minutes at 20uC. Following incubation, DNA was purified using a QIAGEN PCR-clean up kit. Next, we performed dATP addition by incubating 32 mL of DNA with 5 mL 106 NEBuffer 2, 1 mL 10 mM dATP, 3 mL Klenow Exo-minus, and 9 mL nuclease free water at 37uC for 30 minutes. Following incubation, DNA was purified using a QIAGEN MinElute kit to a final volume of 11 mL. Sequencing adapters (custom synthesized by IDT) were ligated to DNA using T/A ligation by incubating 10 mL DNA with 2 mL T4 DNA ligation buffer, 1 mL T4 ligase, 40 mL of 40 mM pre-annealed adapter mix and 6 mL nuclease free water for 15 minutes at 20uC followed by 65uC at 10 minutes to deactivate the DNA ligase. We performed size-selection and PCR amplification as a final step to prepare DNA sequencing libraries. Immediately following ligation, DNA was loaded into a 2%, pre-cast SizeSelect E-Gel (Life Technologies) and run along side a 100 bp ladder. DNA at ,500 bp was removed from the gel into a volume of ,15 mL nuclease free water. To amplify ligated DNA, we performed two replicate PCR reactions for each sample where we used 7.5 mL template DNA, 0.25 mL of 100 mM forward and reverse primers (custom synthesized by IDT), 0.5 mL 10 mM dNTPs, 4 mL 56 High-Fidelity buffer, 0.5 mL Phusion High-Fidelity DNA polymerase, and 5 mL nuclease free water. "