SMRT and Methylome Analysis of ModA

Genomic DNA from each natural modAON and kanamycin knockout pair was prepared using the Qiagen genomic DNA midi kit according to the manufacturer's instructions. SMRT and methylome analysis was carried out as done previously21 (link)22 (link). Briefly, genomic DNA was sheared to an average length of ∼10 kb using g-TUBEs (Covaris, Woburn, MA, USA) and SMRTbell template-sequencing libraries were prepared using sheared DNA. DNA was end repaired, then ligated to hairpin adaptors. Incompletely formed SMRTbell templates were degraded with a combination of Exonuclease III (New England Biolabs; Ipswich, MA, USA) and Exonuclease VII (USB; Cleveland, OH, USA). Primer was annealed and samples were sequenced on the PacBio RS II (Menlo Park, CA, USA) using standard protocols for long insert libraries. Plasmid midi-preps from E. coli cells expressing NTHi 723 ModA2 and a negative control expressing a non-methylase (SiaB), were prepared using the Qiagen plasmid midi kit according to the manufacturer's instructions, and analysed as above.