Smrt bells

The DNA methylomes of gDNA extracted from asynchronous and synchronous cells were obtained by using PacBio SMRT sequencing at SciLife, Uppsala, Sweden. DNA was sheared into 10 kb fragments using a Genemachines HydroShear Instrument (Digilab, Marlborough, MA, USA). SMRT-bells were constructed according to the manufacturer's instructions (Pacific Biosciences, Menlo Park, CA, USA). SMRT-bells longer than 7 kb were selected by BluePippin (Sage Science, Beverly, MA, USA) and sequenced on one SMRT-cell per sample using a Pacific Biosciences RSII sequencer according to the manufacturer's instructions with 4 h movie-time, yielding 221.78x coverage for asynchronous gDNA, 278.33; 289.31; 348.92; 333.91; and 328.97x coverage for synchronous gDNA extracted at T0, T30, T60, T90, and T120 min after release, respectively. DNA modification detection and motif analysis were performed using RS Modification and Motif Analysis.1 method available on PacBio SMRT analysis platform (v 2.3.0). The “motifs.xlsx” files are available in Supplementary Material.

M. thermoacetica was sequenced using a PacBio RSII instrument (Pacific Biosciences). DNA was sheared into 10 kb fragments using a Genemachines HydroShear Instrument (Digilab). SMRT-bells were constructed according to the manufacturer’s instructions (Pacific Biosciences). SMRT-bells were sequenced on two SMRT-cells on a Pacific Biosciences RSII sequencer according to the manufacturer’s instructions with 4 h movie-time. Only reads longer than 10 kb were considered and min QV of 85 considered. The average reference coverage was above ×137, from 49,792 reads, with an average read length of ~11,000 bp.

Genomic DNA was extracted from whole blood using the Qiagen (Hilden, Germany) Puregene Blood Core Kit C. gDNA integrity was assessed with pulsed-field gel electrophoresis 115 ng/well, 17 h runtime at 70 V (Fig. S1). gDNA was sheared with the Diagenode (Liege, Belgium) Megaruptor using long hydropores. A total of 12 µg gDNA was sheared to 60 Kb fragments in a total volume of 300 µl using the preinstalled settings. DNA was concentrated using 0.45× bead/sample ratio of Ampure PB beads and was eluted in 73 µl elution buffer. Qubit dsDNA BR assay was used to quantify DNA concentration.
All libraries were prepared using SMRTbell™ Template Prep Kit 1.0, according to the Procedure & Checklist—Preparing >30 Kb SMRTbells™ (Pacific Biosciences, Menlo Parc, CA, USA). As 10 µg DNA input was used instead of 5 µg, all reaction volumes were doubled until the size-selection step. DNA was sheared using the Megaruptor^®, after which size selection was performed using the BluePippin high-pass DNA size selection with 0.75% DF marker U1 high-pass 30–40 Kb v3 cassette. The range selection mode was set from 25 to 80 Kb. After size selection, Ampure PB bead cleanup steps were performed using 1× bead/sample ratio. DNA damage repair after size selection was performed with the reaction volumes described in the protocol. Qubit dsDNA HS assay was used to quantify DNA concentration.