Isolation and Quantification of Circulating Cell-Free DNA

Genomic DNA was extracted from tumor tissue using the Qiagen Puregene Core kit A (Qiagen) or the QIAamp DNA Mini kit (Qiagen) according to the manufacturer’s instructions and quantified on a Qubit 2.0 fluorometer (Life Technologies). Fragmentation of genomic DNA was achieved by 5 U of AluI or HaeIII restriction enzyme (New England Biolabs) added to each droplet digital PCR (ddPCR) reaction [14 (link)]. Thawed blood and bone marrow plasma, CSF, and urine samples were centrifuged at 2000× g for 5 min to clear debris, then supernatants were centrifuged at 20,000× g for 5 min. Cell-free DNA was purified from a minimum of 40 µL stored samples using the QIAamp Circulating Nucleic Acid kit (Qiagen), then concentrated to 50 μL using the DNA Clean and Concentrator-5 kit (Zymo Research), both according to manufacturers’ directions. Total cfDNA was quantified using the cfDNA ScreenTape assay (Agilent) and Agilent 4200 TapeStation System according to the manufacturer’s instructions [17 (link)]. DNA size distribution was likewise assessed with the Agilent 4200 TapeStation System [17 (link)]. DNA fragments between 100 and 300 bp were considered to be total cfDNA [39 (link)]. Assessment of DNA size distribution and cfDNA quantity does not allow any conclusion to be drawn about the cell(s) of origin.