Extraction and Fragmentation of cffDNA

DNA isolation was performed using the QIAamp DNA Mini isolation kit (Qiagen, Hilden, Germany) from isolated primary AECs following the manufacturer’s instructions. DNA was quantified using a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). To create smaller fragments of cffDNA that would be more like those seen in AF, it was sonicated as previously described [13 (link)]. Non-sonicated “whole DNA” was designated as LF cffDNA and 4-min sonicated DNA was designated as SF cffDNA. cffDNA fragment size of the LF cffDNA and SF cffDNA samples were verified on a 2% agarose gel with ethidium bromide (Invitrogen, Waltham, MA, USA) and visualized under UV transillumination using a Chemidoc (Bio-Rad, Hercules, CA, USA) as previously described [13 (link)].