title: Damage-seq of A549 PCR amplified DNA: BPDE treatment
design: BPDE Treatment GM12878 cells were grown to 700,00 cells/ml in a T75 flask Cells, and A549 cells were grown to ~80% confluence in a 150 mm dish. For damage treatment, the cells were incubated with media containing 25 M of BPDE (#477; MRIGlobal) for 2h. Cells were collected immediately, followed by genomic DNA extraction by DNeasy Blood &amp; Tissue kit  (69504, Qiagen, Hilden, Germany), and quantified by the QuantiFluor dsDNA System (E2670, Promega Corporation, Madison, WI, USA), following manufacturers protocols. For naked DNA samples, DNA was extracted from untreated cells as described above. 3 g of genomic DNA was incubated with 3 M BPDE in a final volume of 30 L at 37 degrees for 2 hours. Treated DNA was purified through a G50 spin column (GE) immediately and subjected to Damage-seq.  Damage-seq Damage-seq was performed as previously described by Hu et al. without biotin purification after the primer extension step. Briefly, Genomic DNA was sheared by sonication with Biotuptor Pico sonicator to generate fragments averaging 300 bp in length.  For PCR-amplified DNA, after sonication and ad1 ligation, 10 ng of the ligation product were PCR-amplified with primers Pu/Pi for 10 cycles. The PCR products were purified and subject to BPDE treatment as described above. Damaged DNA immunoprecipitation was performed with Anti-BPDE Antibody (8E11, Santa-Cruz Biotechnology, sc-52624).  Library quality was assessed using Agilent 4200 TapeStation. Qualified libraries were pooled and sequenced on Illumina NovaSeq 6000 or NextSeq 550 sequencers. Reads were processed following the steps mentioned in Hu et al. Reads containing Ad1 adapters were discarded by cutadapt (version 3.5) and were aligned to hg38 genome using bowtie1 (version 1.3.1). Then, Picard MarkDuplicate (version 2.26.10; http://broadinstitute.github.io/picard) was used to remove read duplicates. Next, unique reads in BED format were further filtered with Bedtools (version v2.27.1) and custom BASH scripts. Reads from replicates of the same condition were merged for further analyses.