Qubit dsdna br assay

Whole-genome bisulfite sequencing single-indexed libraries were generated using NEBNext Ultra DNA library Prep kit for Illumina (New England BioLabs, Ipswich, MA, USA) according to the manufacturer’s instructions with modifications. Five hundred nanograms of genomic DNA was quantified by Qubit dsDNA BR assay (Invitrogen) and fragmented by Covaris S2 sonicator to an average insert size of 350 bp. Size selection was performed using AMPure XP beads and insert sizes of 300–400 bp were isolated. Samples were bisulfite converted after size selection using the EZ DNA Methylation-Gold Kit (Zymo, Irvine, CA, USA) following the manufacturer’s instructions. Amplification was performed after the bisulfite conversion using Kapa Hifi Uracil+ (Kapa Biosystems, Boston, USA) polymerase using the following cycling conditions: 98 °C 45 s followed by 8 cycles of: 98 °C 15 s, 65 °C 30 s, 72 °C 30 s; and a 1-min incubation at 72 °C.
Final libraries were run on 2100 Bioanalyzer (Agilent, Santa Clare, CA, USA) High-Sensitivity DNA assay, samples were also analyzed on Bioanalyzer after shearing and size selection for quality control purposes. Libraries were quantified by qPCR using the Library Quantification Kit for Illumina sequencing platforms (KAPA Biosystems, Boston, MA, USA), using the 7900HT Real Time PCR System (Applied Biosystems, Waltham, MA, USA).

High molecular weight (HMW) was extracted using PacBio’s Nanobind CBB Kit (Pacbio, 102-301-900) from 2 × 10⁶ cells with the Nanobind adherent cultured cells protocol. After extraction, DNA concentration was quantified using the Qubit dsDNA BR assay (Invitrogen, Q32850), sized with a Femto Pulse System (Agilent, M5330AA), and size selected with the PacBio SRE Kit (Pacbio, SKU 102-208-300). Following quality control, the extracted DNA was sheared to a target size of 18–20 kb using the Megaruptor 3 (Diagenode, B060100003). After confirmation of correct sizing, the library preparation was performed SMRTbell prep kit 3.0 (PacBio, 102-141-700) with a PEG wash. The library was sequenced on a Revio flow cell with a 24 h movie time.

Genomic DNA used for alkaline gel electrophoresis was isolated from rnhB_wt strains; RW1628 (dnaE_wild‐type), RW1714 (dnaE_S759C), RW1612 (dnaE_S759N), and RW1610 (dnaE_S759T) and ΔrnhB strains; RW1630 (dnaE_wild‐type), RW1736 (dnaE_S759C), RW1718 (dnaE_S759N), and RW1624 (dnaE_S759T; Table 2), using a previously described method (Ding et al., 2015 (link)). In brief, E. coli from 1.5 ml of overnight culture was pelleted and resuspended in 200 μl of lysis buffer (2% Triton X‐100, 1% SDS, 0.5 M NaCl, 10 mM Tris, and 1 mM EDTA, pH 8.0). Cells were lysed by vortexing in the presence of 0.2 ml glass beads (0.4–0.6 mm diameter) and 200 μl of phenol (pH 7.9) for 2 min, then another min after adding 200 μl of TE buffer. After subsequent extractions with 400 μl of phenol:chloroform:isoamylalcohol (25:24:1) and 400 μl of chloroform, DNA was precipitated with ice‐cold ethanol. DNA was quantified using the Qubit dsDNA BR Assay (Invitrogen), and quantity and quality checked by agarose gel electrophoresis (0.8%, 1× TAE).

Genomic DNA was extracted using the FAST DNA Spin kit (MP Biomedicals, Fisher Scientific, The Netherlands) following the manufacturer’s instructions. We included positive controls, a mock community DNA with known composition [107 (link)] and reagent controls for DNA extraction and PCR. The concentration of genomic DNA was measured fluorometrically using Qubit dsDNA BR assay (Invitrogen). The hypervariable region V5-V6 (~280 bp) of the 16S rRNA gene was amplified with Phusion Hot Start II DNA polymerase (2 U/μL) for 25 cycles using 0.05 μM of each primer (784F–1064R) that both contained sample-specific barcodes at their 5′-end. The amplification program for PCR included an initial step of 98 °C for 30 s, then 25 cycles of at 98 °C for 10 s, followed by an annealing step at 42 °C for 10 s and elongation step at 72 °C for 10 s and a final extension at 72 °C for 7 min. PCR products were purified using MagBio beads according to the manufacturer’s protocol. Purified products were quantified using Qubit dsDNA BR assay kit (Life Technologies, USA) and were pooled in equimolar amounts into one single library. After pooling, the mixed libraries were concentrated using MagBio beads to a concentration needed by the sequencing company. The samples were sequenced on a NovaSeq platform (Illumina) in 2 × 150 base paired-end mode at Novogene (U.K).