Journal: eLife
Article Title: High-resolution mapping demonstrates inhibition of DNA excision repair by transcription factors
doi: 10.7554/eLife.73943
Figure Lengend Snippet: ( A ) NMP-seq methodology. Genomic DNA was first sonicated to short fragments (~400 bp) and ligated to the 1st adaptor (green). After blocking all free 3’-OH groups with terminal transferase and dideoxy-ATP (dd), DNA fragments were digested with AAG and APE1 to generate a new nick with a ligatable 3’-OH group at the NMP damage site. After denaturing to obtain single-stranded DNA, the new 3’ end is ligated to a splint adaptor (2nd adaptor; purple) and the ligation occurs exactly at the damage site. The biotin on the 2nd adaptor allows purification of ligation product with the Streptavidin beads. The purified product is used as the template for PCR amplification, using primers complementary to the 1st and 2nd adaptors. The resulting library is sequenced on an Iron Torrent sequencer using a sequencing primer complementary to the 2nd adaptor. ( B ) NMP-seq read counts in MMS-treated mag1 cells (0.4% MMS for 10 min). (G) and (A) reads are associated with 7meG and 3meA lesions in the genome. ( C ) Schematic for damage mapping in naked yeast genomic DNA. It differs from mapping cellular NMP lesions by inducing damage in purified DNA, instead of cellular DNA bound by proteins. ( D ) NMP-seq read counts for naked genomic DNA treated with 0.4 or 0.2% of MMS for 10 min.
Article Snippet: The NMP lesion site was cleaved by hAAG (NEB, M0313S) and APE1 (NEB, M0282S) to generate a new ligatable 3’ end.
Techniques: Sonication, Blocking Assay, Ligation, Purification, Amplification, Sequencing
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