All the methods were performed in accordance with relevant guidelines and regulations.
Preparation Of Dna
Genomic DNA (gDNA) samples (n = 4) were obtained from the BioBank at the National Center for Child Health and Development. gDNA was extracted from whole blood using the MagCore Genomic DNA Large Volume Whole Blood Kit (RBC Bioscience, MGB1200). DNA samples (200 ng) dissolved in 80 µL of 0.1X TE buffer (pH 8.0) were sheared with Covaris S220 system (Covaris) to the peak-top size of 300 bp.
End Repair, Da-tailing, And Adapter Ligation
All enzymatic reaction mixtures described hereafter were prepared on ice and mixed by pipetting unless otherwise specified. All isothermal incubation and thermal cycling processes were conducted on a thermocycler using PCR eight-tube strips (0.2 mL) and eight-cap strips. Fragmented DNA was filled up to 43 µL with water (Nacalai tesque, 06442-95) and added to 7 µL of NEBNext Ultra II End Prep Reaction Buffer and 3 µL of End Prep Enzyme Mix (NEB, E7120). The mixture was incubated at 20°C for 30 min and at 65°C for 30 min (the heated lid was set to 75°C). Subsequently, 2.5 µL of xGen Methyl UDI-UMI adapter (15 µM; IDT, 10006644), 1 µ of NEBNext Ultra II Ligation Enhancer and 30 µL of Ligation Master Mix (NEB, E7120) were added to the reaction mixture. The mixture (93.5 µL in total) was incubated at 20°C for 60 min (heated lid off). After the ligation reaction, the reaction mixture was purified using 110 µL of NEBNext Sample Purification Beads (NEB, E7120) according to the manufacturer’s instruction (see the Supplementary Text). The adapter-ligated DNA was eluted with 28 µL of Elusion Buffer (NEB, E7120).
Oxidation Of 5-methylcytosines And 5-hydroxymethylcytosines By Tet2
Ten µL of TET2 Reaction Buffer containing TET2 Reaction Buffer Supplement, 1 µL of Fe (II) solution (500 mM, NEB, E7120), was diluted to 4 µM by adding 1249 µl of water. One µL of Oxidation Supplement, 1 µL of DTT, 1 µL of Oxidation Enhancer (T4-BGT) and 4 µL of TET2 (NEB, E7120), and 5 µL of the diluted Fe (II) solution were added to the 28 µL of adapter-ligated DNA. The mixture (50 µL in total) was incubated at 37°C for 60 min (the heated lid set to 45°C). After 1 µL of Stop Reagent (NEB, E7120) was added, the mixture was further incubated at 37°C for 30 min. The reaction mixture was purified using 90 µL of NEBNext Sample Purification Beads (see the Supplementary Text). The TET2-treaded DNA was eluted with 12 µL of water.
Sample Pooling And Hybridization
Twelve µL each of the adapter-ligated and TET2-treated DNA samples were pooled. After 5 µL of Human Cot DNA (IDT, 1080577) was added, the pooled DNA was dried up using a centrifugal concentrator (CC-105, TOMY Digital Biology) for 45 min, and resuspended in 12 µL of water. After 1.25 µL of xGen Universal Blockers TS Mix (IDT, 1075474) was added, the DNA solution (13.25 µL in total) was subjected to the follow three incubation steps: 95°C for 5 min, 65°C for 10 min, and 65°C for 1 min (heated lid set to 105°C). During the first incubation step, 2 µL of 25% SureSelect RNase Block (Agilent Technologies, 5190–9686) was prepared and kept on ice. During the second step, a probe hybridization mixture was prepared by mixing 2 µL of 25% of SureSelect RNAse Block, 6 µL of SureSelect Fast Hybridization Buffer (Agilent Technologies, 5190–9686), and 5 µL of SureSelect XT Human Methyl-Seq Capture Library (biotin-labelled RNA baits) (Agilent Technologies, 5190 − 4661), and kept at room temperature. During the third step, the probe hybridization mixture (13 µL in total) was added to the DNA solution (13.25 µL) on a thermal cycler block. Subsequently, the mixture (26.25 µL in total) was incubated under the following conditions, sixty cycles of 65°C for 1 min and 37°C for 3 sec, for the hybridization of RNA baits to the adapter-ligated and TET2-treated DNA.
Capture And Wash Of Dna/rna Hybrids
The capture and wash of DNA/RNA hybrids were conducted using Dynabeads MyOne Streptavidin T1 magnetic beads (Thermo Fisher Scientific, 65601) and binding/wash buffers in the SureSelect XT HS and XT Low Input kits (Agilent Technologies, 5190–9687) according to the manufacturer’s protocol. Briefly, the hybridized DNA solution was added to the streptavidin beads resuspended in the SureSelect Bindig Buffer and mixed by a plate mixer for 30 min at room temperature. The beads were washed with SureSelect Wash Buffer 1 once at room temperature and subsequently six times with SureSelect Wash Buffer 2 prewarmed at 70 oC. The detailed procedures were described in the Supplementary Text.
Elution Of Dna And Deamination Of Cytosines By Apobec
After completely removing the residual buffer at the final wash procedure, 4 µL of 0.1N NaOH was added to the streptavidin beads in a 0.2 mL PCR tube. The tube was centrifuged briefly for spin-down, mixed by vortex, and centrifuged briefly again. After the tube was placed on a magnetic stand for 1 min, and the supernatant (4 µL), which is expected to contain denatured single-stranded DNA, was transferred to a new PCR tube. Eighty-four µL of water, 10 µL of APOBEC Reaction Buffer, 1 µL of bovine serum albumin (BSA) and 1 µL of APOBEC (NEB, E7120) were added to 4 µL of eluted single-stranded DNA. The mixture (100 µL in total) was incubated at 37°C for 3 h (the heated lid set to 45°C). After this APOBEC reaction, the mixture was purified using 100 µL of NEBNext Sample Purification Beads (see the Supplementary Text) and eluted with 20 µL of water. The eluted DNA was transferred to a new 0.2 mL PCR tube.
Pcr Amplification And Quantification Of Post-capture Library
Quantities of 2.5 µL each of PCR primers (10 µM; 5′-AATGATACGGCGACCACCGA-3′ [P5] and 5′-CAAGCAGAAGACGGCATACGA-3′ [P7]) and 25 µL of NEBNext Q5U Master Mix (NEB, E7120) were added to the eluted DNA (50 µL in total). The mixture was subjected to the following thermal cycling conditions: 98°C for 30 sec, then nine cycles of 98°C for 10 sec, 62°C for 30 sec, and 65°C for 60 sec, and finally 65°C for 5 min. After PCR amplification, the mixture was purified using 45 µL of NEBNext Sample Purification Beads and eluted with 21 µL of Elution Buffer (NEB, E7120). The eluted DNA was transferred to a new tube. The size distribution and the concentration of the amplified capture library were examined using the High Sensitivity Kit (Agilent, 5067 − 4626) and 2100 Bioanalyzer.
Library Quantification And Illumina Sequencing
The final libraries were subjected to paired-end sequencing (151 bp × 2) on a HiSeq-X system (Illumina) at Macrogen Japan Corp. (Tokyo, Japan) with a 20% amount of spike-in PhiX Control v3 Library (Illumina) for colour balancing of low nucleotide diversity. The BCL (base calls) files were converted to fastq files using the bcl2fastq software (Illumina). The sequencing data were analyzed as described in the Supplementary Text.