title: GSM7503618: 12J-naked-DNA; Homo sapiens; OTHER
library_construction_protocol: Genomic DNA was isolated using Sigma GenElute™ Mammalian Genomic DNA Miniprep Kit by following the mannual. CPD-seq 2.0 library was prepared by following our published protocol (Mao and Wyrick, The Nucleus 79–94 (2020), but with some modifications. Briefly, For each time point (e.g., no UV, 0, 6, and 24 h repair), 10 μg of genomic DNA was mixed with 1 ng UV-irradiated pUC19 plasmid DNA. The pUC19 plasmid DNA serves as a spike-in control for library sequencing depths and subsequent repair quantifications. The DNA was incubated with TdT and dideoxy-ATP (ddATP) to block free 3' ends that were generated during DNA isolation step. However, because the genomic DNA was not sheared by sonication, it should contain significantly lower number of nicks and breaks compared to the original CPD-seq protocol. The genomic DNA was treated by the glycosylase T4 endonuclease V (T4 endo V) and AP endonuclease 1 (APE1). T4 endo V is a glycosylase that specifically cleaves a CPD lesion on the 5' side. APE1 further processes the DNA product by T4 endo V and generates a new ligatable 3'-OH group 1 nucleotide upstream of the CPD lesion. After T4 endo V and APE1 digestion, DNA was denatured for single-stranded fragments and the 3'-OH group was immediately ligated to a splint adaptor DNA. After sonication to smaller DNA fragments (~300-400 bp on average), the ligation product was purified with the Streptavidin beads, which recognize the biotin on the adaptor DNA, thus separating CPD fragments from the rest of the genomic DNA. The purified fragments were further processed to add the second adaptor for Illumina sequencing. After PCR amplification for 12 cycles using primers complementary to the two adaptors, the library was sequenced on an Illumina Novaseq platform.