="WGS of endophytic prokaryote culture - " &A2
Roots of adult trees of white poplar were collected in May 2023 in different geographical locations of the European part of Russia. The obtained plant roots were placed in zip-lock bags and transported to the laboratory at +4C for further analysis within 24 h after collection. For isolation of culturable bacteria, the roots were cut into small pieces and sterilized using surfactants and disinfectants: Tween-20 and sodium hypochlorite 3%, after application of which the root was thoroughly washed with autoclaved water. To confirm complete sterilization of the roots surface after disinfection, the roots were incubated in a Petri dish with sterile agarized Luria-Bertani (LB) nutrient medium for three days. In case of colony development on such dishes, the roots were discarded. The roots were then ground in 1 ml of phosphate-salt buffer. The resulting suspension was serially diluted and aseptically poured 0.1 ml into sterile Petri dishes with LB agar medium. Then incubated at 20 C for 48-72 h and colony growth was checked. The pure bacterial strain was obtained by isolation on fresh LB-agar plates. Strains were preserved in 30% (v/v) glycerol at -80 C until further analysis. Before DNA isolation, bacterial samples were stored at -80 C. Total DNA was isolated from bacteria using the DNeasy PowerSoil Pro DNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. DNA was quantified using a Qubit2.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA); quality control was performed on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). The A260/A280 ratio in the DNA samples was 1.8-2.0. Approximately 100 ng of genomic DNA was fragmented into 500-bp-long double-stranded fragments using the Covaris S220 system (Covaris Inc., MA, USA). Double-stranded DNA library was prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NewEngland Biolabs, Ipswich, MA, USA) according to the manufacturer's recommendations. AMPure XP beads were used to select DNA libraries by size (500-600 bp). Sample quantitation at standard concentration dilution to 4 nM was determined using a Qubit2.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and verified on an Agilent 2100 Bioanalyzer using a highly sensitive DNA chip (Agilent Technologies). Genomic DNA of bacteriophage PhiX was used as an internal control. Sequencing was performed using the MiSeq Reagent Kit v3 (300 cycles) on Illumina's MiSeq platform according to the manufacturer's instructions. This resulted in 400 Mb of data (2  150 bp paired-end reads) for each sample. Illumina reads were trimmed from the trailing 3'-end with both fixed quality threshold and sliding window, filtered for average read quality, and adaptors were removed using the trimmomatic 0.39.