Quantifying PCSK9-HBEGF Translocations

The translocation assay targets PCSK9 and HBEGF and was performed as described previously^{86 (link)}. In short, genomic DNA was isolated from HEK293T cells 3 days after transfections using the Gentra Puregene Cell Kit (Qiagen) and was diluted to 10 ng/μL. Custom ddPCR assays were ordered from Bio-Rad detecting balanced translocations between PCSK9 and HBEGF (Supplementary Data 2). AP3B1 (BioRAD) was used as reference assay. ddPCR PCR reaction contained 1× ddPCR Supermix for Probes, no dUPT (Bio-Rad), 1× FAM-labelled HBEGF-PCSK9 custom assay (BioRAD), 1× AP3B1-HEX labelled human reference assay (Bio-Rad), 1/40 HaeIII (Invitrogen) and 50 ng/μL genomic DNA. 20 µL PCR reaction was used to generate lipid droplets with an automated Droplet Generator (Bio-Rad). PCR amplification was performed using the following conditions: 95 °C for 10 min, 40x (94 °C for 30 s, ramp 2 °C/s; 63.2 °C for 1 min) followed by enzyme deactivation at 98 °C for 10 min. Readout was performed with QX 100 Droplet Reader (Bio-Rad) and ddPCR Droplet Reader Oil (Bio-Rad). Data analysis was conducted with QuantaSoft 1.7.4 Software from Bio-Rad.