Exonuclease 3

SMRTbell template libraries were prepared as previously described [28] (link). Two different sized SMRTbell template libraries were employed. Genomic DNA samples were either sheared to an average size of ∼800 base pairs via adaptive focused acoustics (Covaris; Woburn, MA, USA) or to a target size of approximately 8–10 kilobase pairs using Covaris g-TUBEs (Woburn, MA, USA). Fragmented DNA was then end repaired and ligated to hairpin adapters. Incompletely formed SMRTbell templates were digested with a combination of Exonuclease III (New England Biolabs; Ipswich, MA, USA) and Exonuclease VII (Affymetrix; Cleveland, OH, USA). SMRT Sequencing was carried out on the Pacific Biosciences RS (Menlo Park, CA, USA) using C2 chemistry with standard protocols for either small or large insert SMRTbell template libraries.

RASER-FISH was conducted as previously described (Brown et al., 2018 (link)) with minor changes. Briefly, cells were grown on coverslips, labeled for 24 h with BrdU/BrdC mix (3:1) at final conc. of 10 μM, with auxin added at 500 μM for the final 6 h. Cells were fixed in 4% PFA (vol/vol) for 15 min and permeabilised in 0.2% Triton X-100 (vol/vol) for 10 min. Cells were then stained with DAPI (0.5 μg/mL in PBS), exposed to 254 nm wavelength UV light for 15 min, then treated with Exonuclease III (NEB) at 5 U/μL at 37°C for 15 min. Labeled probes (100 ng each) were denatured in hybridization mix at 90°C for 5 min and pre-annealed at 37°C for 10 min. Coverslips were hybridized with prepared probes at 37°C overnight. Following hybridization, coverslips were washed for 30 min twice in 2x SSC at 37°C, once in 1xSSC at RT. Coverslips were blocked in 3% BSA (wt/vol) and digoxigenin was detected with sheep anti-digoxigenin FITC 1/50 (Roche, 11207741910) followed by rabbit anti–sheep FITC 1/100 (Vector Laboratories, FI-6000). Coverslips were stained with DAPI (0.5 μg/mL in PBS), washed with PBS and mounted Vectashield (Vector Laboratories).

Genomic DNA from our enriched triplet sets of S. suis strains LSS89 (modS1 19, 20, and 21 repeats) and SS1056 (modS2 17, 18, and 19 repeats) was prepared from an overnight culture in THB-Y broth, and high-molecular-weight genomic DNA was isolated using the Sigma GenElute kit (Sigma-Aldrich) according to the manufacturer's instructions. SMRT sequencing and methylome analysis was carried out as previously described (48 (link), 49 (link)). Briefly, DNA was sheared to an average length of approximately 5 to 10 kb (genomic DNA) using g-Tubes (Covaris, Woburn, MA), and SMRTbell template sequencing libraries were prepared using sheared DNA. DNA was end repaired, then ligated to hairpin adapters. Incompletely formed SMRTbell templates were degraded with a combination of exonuclease III (New England Biolabs; Ipswich, MA) and exonuclease VII (USB; Cleveland, OH). Primer was annealed and samples were sequenced on the PacBio Sequel system (Menlo Park, CA) using standard protocols for long insert libraries. SMRT sequencing and methylome analysis was carried out at SNPSaurus (University of Oregon, OR, USA).

Genomic DNA from Methanococcus aeolicus PL15/H^p was purified using a Monarch Genome Purification kit (T3010, NEB, Ipswich, MA, USA) and the DNA sample was sheared to an average size of ∼ 10 kb using the G-tube protocol (Covaris, Woburn, MA, USA). DNA libraries were prepared using a SMRTbell express template prep kit 2.0 (100–938–900, Pacific Biosciences, Menlo Park, CA, USA) and ligated with hairpin barcoded adapter lbc1-lbc1. Incompletely formed SMRTbell templates were removed by digestion with a combination of exonuclease III and exonuclease VII (NEB, Ipswich, MA, USA). The qualification and quantification of the SMRTbell libraries were made on a Qubit fluorimeter (Invitrogen, OR) and a 2,100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). SMRT sequencing was performed using an SQ1 (Pacific Biosciences, Menlo Park, CA, USA) based on the multiplex protocol for 10 kb SMRTbell library inserts. Sequencing reads were collected and de novo assembled using the Microbial Assembly version 10.1.0.1119588 program with default quality and read length parameters. In addition to genome assembly (Chin et al., 2013 (link)), the SMRT Analysis pipeline from Pacific Biosciences¹ enables the determination of the epigenetic status of sequenced DNA by identifying the m6A and m4C modified motifs (Flusberg et al., 2010 (link); Clark et al., 2012 (link); Korlach and Turner, 2012 (link)).

Each strain was growth in RB with the appropriate antibiotics (see "Growth media" above) overnight at 37°C with 250 rpm agitation.
gDNA was extracted with the Monarch Genomic DNA purification kit (New England Biolabs; Ipswich, MA, USA) from 1ml of culture.
Libraries from these genomic DNAs were sequenced using the PacBio RSII or Sequel I sequencing platform. Briefly for RSII, SMRTbell libraries were constructed from genomic DNA samples sheared to between 10 and 20 kb using the G-tubes protocol (Covaris; Woburn, MA, USA), end repaired, and ligated to PacBio hairpin adapters. Incompletely formed SMRTbell templates and linear DNAs were digested with a combination of Exonuclease III and Exonuclease VII (New England Biolabs; Ipswich, MA, USA). The SMRTbell library was prepared according to PacBio sample preparation protocol sequenced with C4-P6 chemistry with a 300 min collection time.
For Sequel I libraries, SMRTbell libraries were constructed from genomic DNA samples following the PacBio protocol for Sequel using the kit 100-938-900. DNA qualification and quantification were performed using the Qubit fluorimeter (Invitrogen, Eugene, OR) and 2100 Bioanalyzer (Agilent Technology, Santa Clara, CA). The libraries were prepared for binding following the PacBio guidelines generated by SMRT Link and run on a Sequel I machine.