cfMBD-Seq_4
3 ng of each isolated cfDNA sample was barcoded by using the adapter ligation-based NEXTflex Cell Free DNA-Seq Kit 2.0, until the ligation step with 1:30 diluted adapters and an additional heat inactivation of the ligase for 5 min at 65 C. Afterwards, 90 l of each barcoded sample were pooled and purified with the given sample-to-bead ratio of 1:1 and eluted in 68 l nuclease-free water. 65 l of the eluate were used as input for methyl-CpG-binding domain enrichment with the EpiMark Methylated DNA Enrichment Kit. The protocol was performed according to the instruction manual until the elution step where the sample was eluted with 26 l of nuclease-free water in a thermomixer set at 65 C for 15 min at 1200 rpm. 23 l of the eluate were used for final PCR of the NEXTflex sample prep protocol that was completed according to the manufacturers instructions. Final libraries consisted of a pool of barcoded, methylated cfDNA samples that were sequenced using a  NextSeq 2000 (Illumina).