Methylation experiments:
For the competitive nucleosome methylation experiments, 0.6 pmol of each nucleosome variant were digested with MluI (NEB) for 60 min at 37°C in 10 µL NEB Cutsmart buffer (50 mM KOAc/20 mM Tris-acetate pH 7.9, 10 mM Magnesium Acetate, 100 µg/mL BSA) to remove residual unbound DNA. Afterwards, DNMT3A2 or DNMT3AC was added to the mixture to a final concentration ranging from 0.5 µM to 3 µM and in 80 µL NEB Cutsmart buffer supplemented with 10 mM EDTA and 25 µM AdoMet (Perkin Elmer). The methylation reaction was allowed to proceed for 2 h at 37°C. To stop the reaction and remove all nucleosome-bound proteins, proteinase K was added to the reaction and the sample was incubated for further 60 min at 37°C. The resulting unbound DNA was purified from the reaction mixture using the Nucleospin Gel and PCR cleanup kit (Macherey-Nagel). Bisulfite conversion of the methylated DNA was performed using the EZ DNA Methylation-Lightning kit (Zymo Research). Methylation of free DNA was conducted the same way using 15 µM DNA.
Library preparation and sequencing analysis:
Sample-specific barcodes and indices were added to the DNA by PCR amplification in a two-step PCR process. Briefly, in the first PCR, barcoded primers were used to amplify the bisulfite converted nucleosome DNA using the HotStartTaq Polymerase (Qiagen) and the resulting 321 bp fragment was purified using the Nucleospin Gel and PCR cleanup kit (Macherey-Nagel). In the second PCR step, adaptors and indices required for sequencing were added by amplification with the respective primers and the Phusion polymerase (ThermoFisher). The final 390-bp product was purified and used for Illumina paired end 2x250 bp sequencing. Datasets were analyzed using a local instance of the Galaxy bioinformatics server. Sequence reads were trimmed with the Trim Galore! Tool (developed by Felix Krueger at the Babraham Institute) and subsequently paired using PEAR. The reads were filtered according to the expected DNA length using the Filter FASTQ tool and mapped to the corresponding reference sequence using bwameth to determine the percentage of methylated CpGs.
The naming of the files is described in the Supplemental Table 1 of the accompanying manuscript.
