Methylated DNA Synthesis for Phase Separation

The DNA used for the phase separation assay with different lengths and methylation levels were synthesized by PCR using Q5 polymerase (NEB, M0491S) as described before [72 (link),76 (link)]. In brief, pUC18-MINX plasmid (Table S1) was applied as a template and different reverse (Rev) primers (Table S2) were used to amplify DNA of different lengths. The 800 bp DNA with cytosine methylation was synthesized by replacing the dCTP with dmCTP in the PCR mixture. The 800 bp DNA with CpG methylation was obtained with the CpG methyltransferase M.SssI (NEB, M0226S) after PCR and followed by DNA purification from agarose gel according to the manufacturer’s instructions. Briefly, 1 µg of purified 800 bp DNA product was mixed with 160 µM SAM (S-adenosyl-methionine; NEB, B9003S), methylated by 4 units M.SssI for 4 h at 37°C in the 1 x NEB buffer 2.