Optimized DNA Extraction from FFPE and LBC

Whole blood DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen). DNA from FFPE tissues was obtained from 3 to 6 sections with 10-μm thickness, including more than 30% of the cancer area, or after microdissection using a microdissection laser (Leica Biosystems, Nussloch GmbH, Germany). One mL of residual LBC samples was centrifuged (× 12,000 g), and the pellets were resuspended in 95% ethanol and air-dried for 5 min. Both the FFPE sections and LBC pellets were then incubated with proteinase K (Promega, Madison, WI, USA) for 15 h at 70 °C, followed by a 1-h incubation at 98 °C in lysis buffer (Promega). After centrifugation (× 12,000 g), the supernatants were applied to the Maxwell RSC DNA FFPE kit and Maxwell 16 system (Promega). After measuring the extracted DNA concentration using the Qubit 3.0 dsDNA BR assay kit (Life Technologies, Grand Island, NY, USA), DNA qualities were monitored using the QIAseq DNA quantimize kit (Qiagen). A quality check score (QC score) less than 0.04 was considered as a high-quality DNA, according to our previous report [23 (link)].