DARESOME enables concurrent profiling of multiple DNA modifications in single cells and multi-omics detection of cancer signatures in cell-free DNA

DARESOME enables concurrent profiling of multiple DNA modifications in single cells and multi-omics detection of cancer signatures in cell-free DNA Bulk samples undergo DARESOME protocol and single cells undergo scDARESOME. Cells were lysed and protease treated prior to HpaII digestion. Digested unmethylated sites were ligated with unmethylation-tag (U-tag) adapters. Excess adapters were then removed using NEB Thermolabile USER II enzyme. This was followed by the second round of digestion with MspI and NlaIII enzymes. Hydroxymethylation-tag (H-tag) and Nla-tag (N-tag) adapters were ligated with the digested fragments. Excess adapters and oligonucleotides were removed with Thermolabile USER. 5hmC CCGG sites were glucosylated by glusyl transferese. The samples then undergo MspI digestion, methylated CCGG sites are digested and liagated to Methylated-tag (M-tag). The Read1 and Read2 Illumina sequencing adapters were added through PCR. The amplified library was size selected using BluePippin and quantified using Kapa Library Quantification kit.