Dneasy blood and tissue purification kit

Genomic DNA was purified from U2OS HA-ER-I-PpoI cells using a DNeasy blood and tissue purification kit (Qiagen) according to the manufacturer's instructions. qPCR analysis was performed in a final volume of 10 μL using 10 ng of genomic DNA as a template and 1 pmol of each primer (Table 3). Reactions were carried out using a Roche LightCycler 480 II system for 50 cycles. The purity of the PCR products was determined by melting curve analysis. Cutting efficiency was calculated by measuring the uncut DNA fraction by qPCR using primers spanning the I-PpoI recognition site and comparing that with the amplification of a part of the gene lacking such a site.

Blood samples from patients were plated and single colonies picked and stored at −80 °C until processing. Samples were grown on trypticase soy agar (TSA) (BD, Franklin Lakes, USA) at 37 °C for 24 h, after which DNA was extracted using the Qiagen DNeasy Blood and Tissue Purification kit including a pre-lysis step using lysostaphin for Gram-positive extractions according to the manufacturer’s recommendations (Qiagen, Valencia, CA, USA). DNA was prepared for multiplexed, paired-end sequencing (2×100 bp) on an Illumina GA_IIX instrument using the Library Preparation kit with standard PCR library amplification (KAPA Biosystems, Woburn, MA, USA) as described previously [22 (link)]. Additionally, six samples that failed initial sequencing were resequenced on an Illumina MiSeq instrument using 2×250 V2 technology (Table S1). The average coverage across all 100 isolates was 130×. Genomes were assembled with UGAP (https://github.com/jasonsahl/UGAP), which utilizes SPAdes v3.10.1 [23 (link)] and Pilon [24 (link)] post-assembly error correction. MLST was performed with the raw reads using SRST2 [25 (link)].