Enzymatic methyl-seq

Ten, 50, and 200 ng of NA12878 genomic DNA were combined with 0.1 ng CpG-methylated pUC19 and 2 ng unmethylated lambda control DNA and was made up to 50 µL with 10 mM Tris 0.1 mM EDTA (pH 8.0). The DNA was transferred to a Covaris microTUBE (Covaris) and sheared to 240–290 bp using the Covaris S2 instrument. DNA was sheared twice for 40 sec at duty cycle 10%, intensity 4, and cycles/burst 200. Fifty microliters of sheared material was transferred to a PCR strip tube to begin library construction. NEBNext DNA Ultra II reagents (NEB) were used according to the manufacturer's instructions for end repair, A-tailing, and adaptor ligation of the 0.4-µM EM-seq adaptor (A5mCA5mCT5mCTTT5mC5mC5mCTA5mCA5mCGA5mCG5mCT5mCTT5mC5mCGAT5mC*T and [Phos]GAT5mCGGAAGAG5mCA5mCA5mCGT5mCTGAA5mCT5mC5mCAGT5mCA). The ligated samples were mixed with 110 µL of resuspended NEBNext sample purification beads and cleaned up according to the manufacturer's instructions. The library was eluted in 29 µL of water, and 28 µL of this DNA was oxidized in a 50-µL reaction volume containing 50 mM Tris (pH 8.0), 1 mM DTT, 5 mM sodium-L-ascorbate, 20 mM αKG, 2 mM ATP, 50 mM ammonium iron (II) sulfate hexahydrate, 0.04 mM UDP-glu (NEB), 16 µg TET2, 10 U T4-BGT (NEB). The reaction was initiated by adding Fe (II) solution to a final reaction concentration of 40 µM and then incubated for 1 h at 37°C. Then 0.8 U of Proteinase K (NEB) was added and incubated for 30 min at 37°C. The DNA was purified using 90 µL of resuspended NEBNext sample purification beads according to the manufacturer's instructions. DNA was eluted in 17 µL of water, and 16 µL was then transferred to a new PCR tube and denatured by the addition of 4 µL of formamide (Sigma-Aldrich) and incubation for 10 min at 85°C. The DNA was then deaminated in 50 mM Bis-Tris (pH 6.0), 0.1% Triton X-100, 20 µg BSA (NEB) using 0.2 µg of APOBEC3A (NEB). The reaction was incubated for 3 h at 37°C, and the DNA was purified using 100 µL of resuspended NEBNext sample purification beads according to the manufacturer's protocol. The sample was eluted in 21 µL water, and 20 µL of the DNA was amplified using 1 µM of NEBNext unique dual index primers and 25 µL NEBNext Q5U master mix (NEB M0597) as follows: 30 sec at 98°C; cycling four (200 ng), six (50 ng), and eight (10 ng) times according to DNA input, 10 sec at 98°C, 30 sec at 62°C, and 60 sec at 65°C; with a final extension for 5 min and hold at 4°C. EM-seq libraries were purified using 45 µL of resuspended NEBNext sample purification beads, and the sample was eluted in 21 µL water. Low-input EM-seq libraries for 100-pg to 10-ng genomic DNA inputs were processed as for the 10-ng to 200-ng genomic DNA inputs except 2 U T4-BGT was used. Ten nanograms cfDNA and 10 ng lung FFPE were processed as described for the 10-ng to 200-ng EM-seq libraries, except shearing was not required for the cfDNA. The oxidation reaction for the cfDNA and FFPE DNA reactions was supplemented with 2 mM DTT. Large-insert EM-seq libraries were processed as described for the 10-ng to 200-ng EM-seq libraries, except for the following. The DNA was sheared to 1 kb using a Covaris S2 system with the following settings: duty cycle 5%, intensity 3, cycles per burst 200, and time 40 sec. The clean ups after PCR were as follows: The PCR reaction was either cleaned up as described for the 10-ng to 200-ng EM-seq protocol, or for larger-insert libraries, the volume was increased to 100 µL using water. Sixty-five microliters of resuspended NEBNext sample purification beads was added and the DNA purified according to the manufacturer's protocol. All libraries were quantified using D1000HS tape for TapeStation (Agilent) before Illumina sequencing.