Quantification of DNA Methylation via Bisulfite Sequencing

Bisulfite conversion was done using the EZ DNA Methylation kit from Zymo Research as described before (13 (link)). We converted 200 ng of DNA at 50°C for 12 h from each cell line after 30 days of culturing with ABA. We used primer oVIN-2209 and oVIN-2211 to amplify the converted DNA (Supplementary Material, Table S4). The products were then purified using the NucleoSpin PCR Clean-up kit (Macherey-Nagel). We then performed 2 × 250 bp paired-end MiSeq sequencing (Illumina). The sequencing primers are found in Supplementary Material, Table S4. We processed the reads with TrimGalore (github.com/FelixKrueger/TrimGalore) using -q 20 —length 20 –paired. We aligned the reads using QuasR (62 (link)) to the GFP transgene sequence. We extracted the methylation levels for each CpG in the amplicon with the qMeth() function in QuasR. We calculated the CpG methylation frequencies by dividing the frequency of methylated CpGs by the total number of CpG and expressed it as a percentage.