title: Mycobacterium uberis Genome sequencing
design: The DNA extraction method was used in such a way as to remove as much host DNA from tissue samples as possible. Briefly, host cells were first lysed and host nucleic acids were digested with a benzonase nuclease. Bacterial cells were then lysed using chemical and mechanical disruption, followed by DNA precipitation and purification on silica column. In detail, skin samples (50-200 mg) in 70% ethanol were first rehydrated in Hanks balanced solution (Thermo Fisher Scientific, USA, MA) prior to mincing with scissors. Cells were detached from the tissue by 30 min incubation at 37C with a mixture of 0.5 U of collagenase and dispase (Roche Diagnostics, Mannheim, Germany), followed by incubation at 56C with 10 mg/ml of trypsin (Sigma-Aldrich, St. Louis, Missouri, USA) until complete digestion. Free cells were then resuspended in 1 mL of phosphate-buffered saline (PBS) and DNA was extracted using the QIAmp DNA microbiome extraction kit (Qiagen, Hilden, Germany) according to the manufacturers recommendations. DNA samples were sonicated to obtain 500 bp long fragments with the S220 Covaris (Covaris) using the manufacturer's protocol. 50 l of each extract were used for indexed library preparation following the protocols described below and 2 l were used for quantification using the Qubit dsDNA HS Assay Kit and the Qubit 2.0 Fluorometer (Thermo Fisher Sc., USA, MA). Sequencing libraries were synthesized with the Kapa Hyper prep kit (Kapa Biosystems, Wilmington, MA, USA), according to the manufacturers instructions. After the final amplification step, libraries were then quantified using Qubit dsDNA BR Assay Kit (Thermo Fisher Sc., USA, MA) and the fragment size was assessed on a Fragment Analyzer (Advanced Analytical Technologies, Inc. Ankeny, USA). Finally, libraries were multiplexed and sequenced using single-end reads on an Illumina HiSEq 2500 instrument.