GSM7047544: NHF1_UVC0h; Homo sapiens; OTHER
NHF1 or primary melanocyte genomic DNA was isolated with GenElute Mammalian Genomic DNA Miniprep kits (G1N70, Sigma), following the manufacturer's manual. Cells used for naked DNA samples were cultured and DNA isolated as previously described, excluding the UV exposure. Following genomic DNA isolation, samples were pipetted onto a microscope cover slip in 10µL aliquots, and exposed to UV irradiation. The genomic DNA from NHF1 cells were exposed to either 80J/m2 UVC or 500J/m2 UVB and the DNA from primary melanocytes were exposed to 400J/m2 UVC or 2500J/m2 UVB. Library preparation then continued as described, resuming at sonication. CPD-capture-seq procedure is adopted from the published emRiboSeq method (Nature Protocols 2015 10, 1433-1444) and our recently published CPD-seq protocol (Methods in Molecular Biology 2020, 2175, 79-94). Briefly, sonicated DNA fragments were ligated to a double-stranded adaptor DNA F1, and free 3'-OHs were blocked by  terminal transferase and dideoxy-GTP.  CPD lesions were cleaved with T4 endonuclease V and apurinic/apyrimidinic (AP) endonuclease, APE 1, to generage new 3'-OHs. DNA was then ligated to biotin labelled second adapter, S2.   The single stranded DNA was purified with streptavidin beads and final PCR was done for upto 6 cycles with F1 primer and different barcoded RAPID primers ( RAPID 1:TAGAACAC; RAPID 2: GAGCCAAT; RAPID3: CAGATCTG; RAPID 4:TGTGAAGA; RAPID 5:ACAGTGGT; RAPID 6:TGAACTGG; RAPID 7:TCTACGAC; RAPID 8:GCAATCCG; RAPID9:TCCGTCTT; RAPID 10:TCAGGAGG) to generate libraries for different  samples. The resulting DNA libraries were used for DNA sequencing. CPD-seq library fragments were captured for selected genomic regions (~4000 regions, including recurrent mutations in melanoma, ETS binding sites, etc.) by the company RAPID Genomics prior to sequencing.