Invitrogen chargeswitch gdna mini tissue kit

L4 stage animals were irradiated with doses of 20 Gy, 40 Gy, 60 Gy and 80 Gy using a Cs-137 source (IBL 437C, CIS Bio International), and doses of 50 J, 100 J, 200 J and 500 J using a Waldman UV 236 B device with a spectrum of 280 nm to 360 nm (UV-B / UV-A light). cisplatin exposure was performed as described [27 (link)]. In short, animals were incubated under gentle shaking for 16h at 20°C in M9 liquid culture with cisplatin (Sigma-Aldrich, P4394) concentrations from 0 to 400 μM (S1 Table). Treated animals were allowed to recover and 3 times 3 adults per genotype and dose were transferred onto fresh plates 24h post treatment. Adults were removed after 4h and eggs laid within this time were allowed to hatch and scored for viability [26 (link), 27 (link)]. 2 F1 L4s per plate were then singled. For higher doses of radiation, not all F1 animals produced progeny. Progeny of only one of each set of 2 F1 lines was frozen, providing 3 independent lines per genotype and treatment. Genomic DNA was isolated using Invitrogen ChargeSwitch® gDNA Mini Tissue Kit (Thermo Fisher Scientific, CS11204) and sent for sequencing.

All C. elegans strains used in this study, newly and previously generated [10 (link)–12 (link)] (S1 Table) were backcrossed 6 times against the wild-type N2 Bristol reference strain TG1813 [10 (link)–12 (link)], (Fig 1A). The majority of strains were clonally propagated for 20 or 40 generations as described previously [10 (link)]. rtel-1, smc-5, smc-6 lines were grown for 5 generations as these lines tended to become sterile when grown for more generations (S1 Table for number of lines and generations per genotype). As described in detail [10 (link)] 5–10 single L4 stage hermaphrodites (F1s) were randomly chosen for each genotype and transferred to individual 1× NGM plates seeded with OP50 bacteria. Every 3–4 days, 1 single L4 hermaphrodite was randomly chosen among the progeny per plate and individually propagated further, a process repeated until the indicated generation (F5, F20 and F40). Once final generation hermaphrodites had produced clonal progeny 5 lines were transferred to 9 cm 3× NGM plates and allowed to reach starvation. Mixed stage worms (the majority of which two generations the final transferred L4) were washed off plates, washed 3× in M9 medium, pelleted and frozen in liquid nitrogen [10 (link)]. Genomic DNA was isolated from three samples using the Invitrogen ChargeSwitch® gDNA Mini Tissue Kit (Thermo Fisher Scientific, CS11204) [12 (link)].