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. Author manuscript; available in PMC: 2014 May 23.
Published in final edited form as: Cell. 2013 May 23;153(5):988–999. doi: 10.1016/j.cell.2013.04.033

Figure 3. ssDNA-Seq approach and validation.

Figure 3

(A) During ssDNA-Seq ssDNA is stabilized in live cells by treatment with KMnO4, which selectively oxidizes exposed thymidines. Cordycepin and TdT are used to block pre-existing 3’ DNA free ends and ssDNA is digested with mung bean nuclease. DNA ends exposed by nuclease treatment are then biotinylated and, following sonication, streptavidin selected and deep sequenced. (B) Distribution of ssDNA signals in Raji cells. +5Kb and −5Kb represent ssDNA signals aligning within 5Kb upstream of TSS or downstream of gene stop codons respectively. Numbers in parenthesis are expected percentages if reads were randomly distributed. (C) Composite diagram showing ssDNA and PolII signals around TSSs of human genes in Raji cells. (D) Composite diagrams showing the distribution of 5’ (blue) and 3’ (red) ssDNA-Seq tags around SIDDs predicted for the mouse genome. (E) Deep-sequencing profiles at mouse SIDDs obtained with anti-PolII antibodies. (F) ssDNA-Seq analyses of relaxed (upper) or supercoiled (lower) pFLIP plasmids containing human Myc FUSE element. ssDNA-Seq 5’ and 3’ tags are represented in blue and red respectively.