Extended Data Fig. 1: Drawbacks of previous psoralen-based supercoiling assays (A-F) and the performance of ATMP-seq in supercoiling measurement (G-J). | Nature Structural & Molecular Biology

Extended Data Fig. 1: Drawbacks of previous psoralen-based supercoiling assays (A-F) and the performance of ATMP-seq in supercoiling measurement (G-J).

From: Topoisomerase-modulated genome-wide DNA supercoiling domains colocalize with nuclear compartments and regulate human gene expression

Extended Data Fig. 1

(A) Supercoiling-sensitivity of the TMP-DNA reaction. Crosslinking probability was reported based on experiments using an 8-kb plasmid (two biological replicates). (B) Top panel: Human genome coverage of non-crosslinked gDNA fragments remaining after three rounds of denaturation and exonuclease digestion, at 100-kb resolution, leading to genomic bias. Bottom panel: Human genome coverage of gDNA, suggesting the genomic bias of remaining non-crosslinked gDNA fragments in the top panel cannot be explained by gDNA sequencing data. (C) Schematic of TMP reverse-crosslinking, leading to three types of products including reverse-crosslinked (orange), remain-crosslinked (green) and DNA-damaged (blue). (D) Background noise revealed by analyzing the products of TMP reverse-crosslinking by 254-nm UV illumination for 1-5 min. (E) Background noise revealed by analyzing the products of TMP reverse-crosslinking by various conditions of alkaline treatment. Condition 1: 175 mM NaOH and 1.5 mM NaCl, 65 °C incubation for 3 h. Condition 2: 100 mM NaOH, 75 °C incubation for 30 min. Condition 3: 175 mM Na3PO4 and 1.5 mM NaCl, 65 °C incubation for 3 h. Condition 4: 100 mM Na3PO4, 70 °C incubation for 30 min. (F) Background noise revealed by analyzing the products of TMP reverse-crosslinking by 95% formamide and 10 mM EDTA, 90 °C incubation for 10-20 min. (G) ATMP monoadduct density upon reaction with supercoiling-relaxed DNA wrapped by nucleosomes with various densities. Nucleosome density was reported as the percentage of DNA bases wrapped by the nucleosome (two biological replicates). (H) Workflow of ATMP-seq’s sample and control to normalize DNA sequence bias. (I) Technical reproducibility of the ATMP-seq assay at 100-kb resolution, when sequenced by 60 million Illumina reads. (J) Correlation between ATMP-seq’s technical duplicates at 100-kb resolution, when sequenced by 60 million Illumina reads.

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