GSM8595233: csb-1 L1 5 min CPD XR-seq Rep2; Caenorhabditis elegans; OTHER
L1 stage worms were irradiated with 4 kJ/m2 UVB at collected in M9 buffer at indicated repair times. Worms were washed 3 times with M9 buffer and then, the pelleted worms were then incubated for 2 h at 62 °C with 450 μl of Worm Hirt Lysis Buffer (0.15M Tris pH 8.5, 0.1M NaCl, 5mM EDTA, 1% SDS) and 20 μl of Proteinase K (NEB, cat. no. P8107S). Subsequently, 120 μl of 5M NaCl was added, and the mixture was inverted to ensure proper mixing, followed by an overnight incubation and one hour centrifugation at 4 °C. Supernatant was treated with Ribonuclease A (Sigma-Aldrich, cat. no. R4642) and Proteinase K. Next, DNA is precipitated using phenol-chloroform extraction and ethanol precipitation, resolved in TE and incubated with anti-(6-4)PP (Cosmo Bio, cat. no. CAC-NM-DND-002) or anti-CPD (Cosmo Bio, cat. no. CAC-NM-DND-001), antibodies at 4°C overnight. Purified excised oligonucleotide containing either (6-4)PP or CPDs were processed for XR-seq libraries. Purified excised oligomers were ligated with adaptors. Ligation products were subjected to immunoprecipitation with anti-CPD or anti-(6-4)PP antibodies. Purified DNA were repaired by (6-4)PP photolyase or CPD photolyase. Repaired DNA were PCR-amplified by Kapa Hifi HotStart ReadyMix with RP1 and RPIn (n means different indexsequences, compatible with Illumina TrueSeq small RNA kit) for 15 cycles. The PCR products were  subjected to electrophoresis in a 10% native polyacrylamide gel in 1x TBE. Gel slices corresponding to 130-to 155-nt fragments were excised. The DNA was eluted. Libraries were pooled together and sequenced in a NextSeq 2000 platform.