GSM8595186: WT L1 48 hrs (6-4)PP Damage-seq Rep1; 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, genomic DNAs were isolated with QIamp DNA Mini Kit/tissue protocol. Ultrasonic fragmented genomic DNAs were purified using an equal volume of HighPrep PCR beads (MagBio). Purified DNA (~1 µg) was used for end-repair and dA-tailing and adaptor ligation (NEBNext Ultra II DNA Library Prep Kit) following manufacturer's instructions. Damage-containing DNA fragments were purified with either anti-(6-4)PP (Cosmo Bio, cat. no. CAC-NM-DND-002) or Anti-CPD (Cosmo Bio, cat. no. CAC-NM-DND-001). The purified DNA was primer extended in the presence of 30 pmol Bio3U (biotin elongation primer, 5′-bio-AGAGTG/dU/GACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′) using NEBNext Q5 Hot Start HiFi PCR Master Mix. Next, undamaged DNA strands were captured by 20 pmol (2 µL) of SH oligo (SH, Subtractive hybridization, 5′-bio-NNGACTGGTTCCAATTGAAAGTGCTCTTCCG-SpC-3′), DNAs were purified using phenol-chloroform extraction and ethanol precipitation. The DNA was then ligated to a second adaptor ligation using T4 DNA ligase HC (Thermo) at 16 °C overnight. After quality check, the DNA samples were purified with HighPrep PCR beads, and ligated DNAs were PCR amplified by NEBNext Ultra II PCR Master Mix with NEBNext Multiplex Oligos for Illumina (New England Biolabs). The PCR libraries were purified with HighPrep PCR beads. Libraries were sequenced by an Illumina NextSeq2000P2 with 50-bp.