Nanophotometer np80

Singular bacteria colonies were picked from CSM agar (pH 6.5) and incubated in 20 mL Tryptic Soy Broth medium in 150 mL baffled shaking flasks at 120 rpm and 28°C overnight. HMW gDNA was extracted according to the instructions of the Quick‐DNA HMW MagBead Kit (Zymo Research).
To assess the quantity and purity of the obtained DNA, 260/280 nm absorption ratios and concentrations were measured with a photometer (Nano Photometer NP80; IMPLEN) and a Qubit 4 fluorometer with the Qubit 1X dsDNA HS Assay‐Kit (Thermo Fisher Scientific). To confirm the high molarity of the gDNA, fragment sizes were analyzed with a Femto Pulse capillary electrophoresis instrument (Agilent Technologies).
When samples passed the quality control, shearing of 8 µg gDNA in 150 µL Elution Buffer was conducted with g‐TUBEs (Covaris), utilizing 1700g in a tabletop centrifuge. This yielded DNA fragments with a size of ca. 12 kbp, as confirmed with Femto Pulse. Subsequently, HiFi libraries were prepared according to the SMRTbell prep kit 3.0 manual, fusing barcoded adapters to the samples (Pacific Biosciences). Libraries were stored at −20°C until the day of sequencing, where primers and the polymerase bound the samples with the Sequel II Binding Kit 3.2 (Pacific Biosciences), closely following the manufacturer's recommendations.

Bacterial strains B. pumilus 7P (soil isolate, WT) and B. pumilus 3-19 (a strain with resistance to streptomycin) were used. The 7P strain was isolated from the soil of the Republic of Tatarstan (Russia) and identified on its ability to produce ribonuclease (binase). Both 7P and 3-19 strains were used for genomic DNA isolation and sequencing library preparation.
B. pumilus cultures were incubated at 37 °C for 14 h in an aerobic atmosphere. For bacterial cultivation, LB (Lysogeny broth and Lysogeny agar) medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 8.5) was used. High molecular weight bacterial DNA was extracted using phenol/chloroform method [19 (link)]. The cultures of B. pumilus 7P and 3-19 strains were grown up to OD₆₀₀ = 1.0. The cell pellet was resuspended in TEN buffer (10 mM Tris-HCl, pH 8.0; 10 mM EDTA; 150 mM NaCl) and for cleaving of bacterial cell wall lysozyme was added. RNase (20 mg/mL), SDS (10%), and proteinase K (20 mg/mL) were used for the enzymatic digestion of proteins and nonnucleic acid cellular components. DNA extraction was performed using phenol and a mixture of chloroform:isoamyl alcohol (24:1). The aqueous phase was transferred into ice-cold ethanol and stored at 4 °C. The quantity and quality of the purified DNA were measured by NanoPhotometer NP80 (Implen, Westlake Village, CA, USA) and by electrophoresis on a 1.5% agarose gel, respectively.

Total RNA was isolated from the samples of interest using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following standard protocol. RNA quality was assessed using a spectrophotometer (NANOPhotometer^® NP80, IMPLEN, Westlake Village, CA, USA).