Kapa hifi uracil readymix

Glycerol stocks (1 μL) were used to inoculate 5 mL lysogenic broth containing the standard supplements. Cultures were shaken at 250 rpm at 37 °C for 15 hours. From the overnight culture, 1 mL was transferred into a 500 mL shake flask containing 150 mL lysogenic broth with the standard supplements and shaken at 250 rpm at 37°C. The cultures were supplemented with 0.0175% w/v arabinose and 1mM IPTG at an optical density of 0.3, and shaken for 4 hours at 250 rpm at 37°C. A 5 mL aliquot of the culture was centrifuged, and plasmid DNA was extracted from the pelleted cells using the Plasmid Miniprep Kit. Preparation of the purified DNA for high-throughput bisulfite sequencing was as described [13 (link)]. Briefly, DNA was sheared to 300 bp (Diagenode Bioruptor Pico), then end repaired and methylated adaptors ligated using NEBNext Ultra (NEB). Bisulfite treatment was performed with EZ Methylation Lightning (Zymo) then amplified with Kapa Hifi Uracil+ ReadyMix from Kapa Biosystems and NEBNext Multiplex Oligos for Illumina (Methylated Adaptor) from NEB for 8 cycles. DNA concentration was determined using qPCR (Kapa Illumina Library Quantification Kit), and size distribution was confirmed using the High Sensitivity Bioanalyzer. Libraries were subsequently sequenced on an Illumina MiSeq using v3 chemistry. Data was deposited in SRA under Bioproject PRJNA503938.