title: WGS of Strix occidentalis caurina: adult female blood
design: We isolated DNA using a Gentra Puregene Kit (Qiagen) and used a Hydroshear DNA Shearing Device (GeneMachines) to shear 25 µg in DNA in 100 µL volume with 30 cycles of shearing using speed code 3. We checked the sheared DNA on a 1% agarose gel and saw that fragments had been sheared between 400-1000 bp. We additionally mechanically sheared the DNA by performing 15 passes through a 28 gauge x 1/2 inch needle attached to a 1 cc U-100 Insulin Syringe (Becton, Dickinson and Company). We performed end-repair using 4266 ng sheared DNA in an End-It DNA End-Repair Kit (Epicentre). We incubated the reaction at room temperature for 45 minutes and then inactivated the enzyme by heating to 72°C for 10 minutes followed by cleaning with a DNA Clean & Concentrator -5 kit (Zymo Research). We then added 3' A tails in a reaction with 2 µL 10X NEBuffer 2, 0.5 µL 100 mM dATP (Invitrogen), 1 µL Klenow Fragment (3'→5' exo-) (NEB), and 16.5 µL cleaned end-repaired product. We incubated for 45 min at 37°C and then 20 min at 75°C to inactivate the enzyme. We cleaned the reaction with a DNA Clean & Concentrator -5 kit (Zymo Research). We then ligated Illumina-compatible adapters using 1 µL 10X Fast-Link Ligation Buffer (Epicentre), 1 µL 10 mM ATP (Epicentre), 5 µL of end-repaired DNA (0.7835 µg), 2 µL of annealed Illumina-compatible adapters at 10 µM (Integrated DNA Technologies), and 1 µL Fast-Link DNA Ligase (Epicentre) for 10 µL total reaction volume. We incubated the ligation reaction overnight at 16°C and then used 1.5X Agencourt AMPure XP (Beckman Coulter) magnetic beads to clean the ligase reaction and remove any extra adapters. We performed a PCR using 10 µL Klentaq LA 10X Buffer with MgCl (Sigma-Aldrich), 2 µL 12.5 µM dNTPs, 2 µL each of two Illumina-adapter-compatible primers at 10 µM, 2 µL KlenTaq LA DNA Polymerase Mix (Sigma-Aldrich), half of the cleaned ligase reaction in 10 µL, and water to make a 100 µL reaction volume. We ran the PCR in two 50 µL aliquots at 94°C for 5 min; then 2 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 68°C for 3 min; and we performed a final extension at 68°C for 5 min. We removed the PCR products after the final extension and and then cleaned them using a DNA Clean & Concentrator -5 kit (Zymo Research). We used a LabChip XT DNA 750 Assay Kit on a LabChip XT (PerkinElmer) automated nucleic acid fractionation system to select library fragments in the size range of 600-700 bp. We performed a final PCR using 5 µL Klentaq LA 10X Buffer with MgCl (Sigma-Aldrich), 1 µL 12.5 µM dNTPs, 1 µL each of two Illumina-adapter-compatible primers at 10 µM, 1 µL KlenTaq LA DNA Polymerase Mix (Sigma-Aldrich), 5 µL library off of LabChip, and water to make a 50 µL reaction volume. We ran the PCR at 94°C for 2 min; then 17 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 3 min; and we performed a final extension at 72°C for 5 min. We removed the PCR products after the final extension and and then cleaned them using a DNA Clean & Concentrator -5 kit (Zymo Research). We pooled the final library with two other libraries equimolarly and we obtained 350 bp read 1 and 250 bp read 2 for a total of 600 bp of paired-end read data from one lane (approximately 1/3 of one lane for this library) using a 600-cycle MiSeq Reagent Kit v3 on a MiSeq (Illumina).