DMN-seq input of Lambda DNA hypomethylation enrichment
Lambda DNA-only samples were prepared by fragmenting 200 ng -DNA using NEBNext dsDNA Fragmentase, followed by purification and spike-in with 1% each of 200 bp and 164 bp dsDNA oligos. Samples underwent end-repair/dA-tailing, adapter ligation using either methylated NEBNext or customized phosphorothioate-modified DME adaptors, and subsequent purification with AMPure XP beads. The DME adaptor-ligated samples were then subjected to DME enzyme treatment, multiple exonuclease digestions to enrich for 5-phosphorylated fragments, and TACS ligation for library preparation. Finally, libraries were PCR-amplified, purified, quality-checked, and sequenced using the Illumina NextSeq 1000/2000 platform.