Mapping DNA Methylation via µWGBS

Sequencing libraries for DNA methylation mapping were prepared using the µWGBS protocol^{24 (link)}. Starting directly from lysed cells in digestion buffer, proteinase K digestion was performed at 50 °C for 20 minutes. Custom-designed methylated and unmethylated oligonucleotides were added at a concentration of 0.1% to serve as spike-in controls for monitoring bisulfite conversion efficiency. Bisulfite conversion was performed using the EZ DNA Methylation-Direct Kit (Zymo Research, D5020) according to the manufacturer’s protocol, with the modification of eluting the DNA in only 9 µl of elution buffer. Bisulfite-converted DNA was used for single-stranded library preparation using the EpiGnome Methyl-Seq kit (Epicentre, EGMK81312) with the described modifications. Quality control of the final library was performed by measuring DNA concentrations using the Qubit dsDNA HS assay (Life Technologies, Q32851) on Qubit 2.0 Fluorometer (Life Technologies, Q32866) and by determining library fragment sizes with the Agilent High Sensitivity DNA Analysis kit (Agilent, 5067-4626) on Agilent 2100 Bioanalyzer Station (Agilent, G2939AA). All libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the 2x75bp paired-end setup on the Illumina HiSeq 3000/4000 platform.