Mapping CPD formation with modified CPD-seq in A375 naked DNA treated with UVC
THIS IS EXTENDED SUBMISSION FOR THE STUDY PRJEB57327 AT ENA.  Genomic DNA Extraction protocol:  Genomic DNA was extracted with QIAgen DNA blood mini kit CPDs were mapped with a modified CPD seq method (Mao et al 2016). Purified DNA 12 ug was sonicated to 400 bp and size selected with SPRI select beads (Life Technologies) and the purified product (approx. 4 ug) was subjected to NEBNext end repair and NEBNext dA-tailing modules (NEB). ARC141/142 was then ligated to the sheared and repaired ends O/N with NEBNext Quick Ligation module. DNA was purified with CleanPCR beads and treated with Terminal Transferase (TdT, NEB) and dideoxy ATP (Roche) for 2h at 37 degrees. DNA was purified and incubated with 30 units T4 endonuclease V (NEB) at 37 degrees for 2 h, followed by purification and treatment with APE1 (NEB) at 37 degrees for 1.5 hr. DNA was purified and treated with rSAP (NEB) 37 degrees 1 hr followed by deactivation at 65 degrees for 15 minutes. DNA was purified, denatured at 95 degrees for 5 min, cooled on ice and ligated with the biotin-tagged “ARC double” overnight at 16 degrees with NEBNext quick ligation module. DNA fragments with the biotin tag were captured with Streptavidin Dynabeads (Invitrogen) and the DNA strand without the biotin label was released with 0.15 M NaOH. This single stranded DNA was used as the template to synthesise double stranded products using ARC153. The now double stranded library was purified and amplified with ARC140 and ARC78-82 to add Illumina barcodes and indexes.  Data processing: Library strategy: CPD-seq  Alignment with Bowtie 2 version 2.3.1, standard parameters. For UV treated A375 naked DNA, standard parameters Duplicates were marked with Picard MarkDuplicates version 2.18.7, with the parameter VALIDATION_STRINGENCY=LENIENT. Non-duplicate R1 reads belonging to proper pairs were imported into R and further processed with Bioconductor packages. The 2 bases upstream and on the opposite strand of each read on R1 were saved as CPDs. The DNA sequence of each CPD was obtained with the R Bioconductor package BSgenome's getSeq function and non-dipyrimidine "CPDs" were discarded.   Genome_build: hg19  Supplementary_files_format_and_content: Bed. The files contain the positions of all CPDs detected by sequencing, with optical duplicates and non-dipyrimidine "CPDs" removed. Duplicated positions indicate that more than one CPD was detected at that position. Due to the low coverage, we found it easier to work with files with each line representing a CPD, rather than using scores to indicate the number of CPDs at any given position.