Rnase t1

For mass spectrometry analysis, ∼500 ng of tRNA^Phe(GAA) were digested with 100 units of RNase T1 (Sigma) in a final volume of 10 μl at 37°C for 4 h. RNase T1 cleaves the phosphodiester bond between the 3′-guanylic residue and the 5′-OH residue of adjacent nucleotides and generates 3′-phosphate nucleosides. One microliter of digest was mixed with 9 μl HPA (40 mg/ml in water:acetonitrile 50:50) and 1 μl of the mixture was spotted on the MALDI plate and air-dried (‘dried droplet’ method). MALDI-TOF MS analyses were performed directly on the digestion products using an UltrafleXtreme spectrometer (Bruker Daltonique, France). Acquisitions were performed in positive ion mode.

Age-synchronised L4 worms, grown under different BaP concentrations, were collected from plates with M9 buffer, serially washed with M9 buffer, mixed with sterile glass beads, and flash-frozen in liquid nitrogen. A 400 μL mixture of ethylenediaminetetraacetic acid (EDTA, 1 mM) and tris(hydroxymethyl)aminomethane (Tris, 50 mM) was added to each tube before vertexing for 5 min. Then, the supernatants were transferred to new 1.5-mL microcentrifuge tubes, and 9 μL of an RNase mixture of equal amounts of pancreatic ribonuclease (RNase A, 10 mg/mL, Sigma-Aldrich) and ribonuclease T1 (RNase T1, 50 KU, Sigma-Aldrich) were added to the samples. The tubes were incubated on a shaker (400 rpm) at 37 °C for 30 min. Thereafter, 40 μL of a freshly prepared proteinase K [10 mg/mL dissolved in a mix of EDTA (1 mM) and Tris (50 mM)] were added and mixed with the samples. The tubes were incubated on a shaker (400 rpm) at 37 °C overnight. The following day, DNA was isolated using a standard phenol–chloroform extraction method. DNA pellet was resuspended in Tris-EDTA (TE) buffer were stored at − 20 °C until analysis. The presence of BaP-derived DNA adducts (dG-N²-BPDE) was assessed using the nuclease P1 enrichment method of the ³²P-postlabelling protocol as described previously (Arlt et al. 2008 (link); Phillips and Arlt 2020 ).