Qubit dsdna br

Bacterial DNA was extracted using the UltraClean microbial DNA isolation kit (MO BIO Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions. DNA concentrations were measured using a Qubit fluorometer (Life Technologies, Waltham, MA, USA) and the Qubit dsDNA BR and the Qubit dsDNA HS assay kits (Life Technologies). A DNA library was prepared using Nextera XT DNA library preparation kit with the Nextera XT index kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Library fragment lengths were assessed on a 2100 Bioanalyzer using the high sensitivity DNA kit (Agilent Technologies, Santa Clara, CA, USA), and the library was subsequently sequenced on a MiSeq sequencer (Illumina) using the MiSeq reagent kit v3, 600-cycle (Illumina). Quality trimming of reads was performed using the FastQ toolkit v 2.0.0 on the Illumina BaseSpace server (BaseSpace Labs) with a Q threshold set to ≥20. De novo genome assembly of paired-end reads was performed using SPAdes Genome Assembler v 3.6.0 default settings [25 (link)]. A minimum coverage >30-fold and N50 >50,000 was considered acceptable.
Raw sequencing reads was deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA558173.

C10 cells were exposed to B[a]P (0.3, 1 and 3 µM), 10 µM of the binary PAH mix, or the combination of 1 µM B[a]P + PAH mix (0.01, 0.1, 1, 5 or 10 µM, depending on the experiments) for 24 h in three separate experiments. Untreated and DMSO-treated cells were included as negative controls. Cells were harvested and their pellets stored at − 80 °C. DNA was isolated from individual samples (5-mL aliquots, each containing ~ 9–16 × 10⁶ cells) using the QIAamp® DNA Blood Maxi Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The first two eluates with nucleic acids were pooled and treated at 37 °C with DNase-free RNase A (Qiagen, Hilden, Germany; final concentration 20 µg/mL) and RNase T1 (Sigma-Aldrich, Taufkirchen, Germany; final concentration 10 µg/mL) in TE buffer (10 mM Tris–HCl, 1 mM Na2-EDTA; pH 8.0). After 30-min incubation, NaCl (final concentration 100 mM) and two volumes of 95% ethanol were added. Samples were vigorously mixed and allowed to stand at RT for 10 min prior to centrifugation (13,000g, 2 min, RT). Pelleted DNA was dissolved in ultrapure water. Specific content of DNA and remains of RNA were determined using Qubit® dsDNA BR and Qubit® RNA HS assay kits with a Qubit® 3.0 fluorimeter according to the manufacturer’s instructions (Life Technologies, Darmstadt, Germany). The remaining RNA content was ≤ 5%.

Single-indexed cDNA libraries were generated using the SMARTer Stranded RNA-Seq Kit (Clontech Inc.) in accordance with the manufacturer’s instructions. Fluorescent measurement of DNA concentrations in each library was performed using Qubit dsDNA BR and ssDNA assay kit (Thermo Fischer Scientific).