Fig. 1: Overview of the iMUT-seq technique. | Nature Communications

Fig. 1: Overview of the iMUT-seq technique.

From: iMUT-seq: high-resolution DSB-induced mutation profiling reveals prevalent homologous-recombination dependent mutagenesis

Fig. 1

A Experimental design of iMUT-seq, first Damage-Induced via AsiSI (DIvA) cells are treated with hydroxytamoxifen (OHT) to translocate the AsiSI-ER-AID fusion protein to the nucleus where it generates DSBs. Next, the fusion protein is degraded by activating it’s auxin-inducible degron (AID) with auxin (IAA) treatment, allowing the cell to fully repair all the DSBs. B Western blot of AID-DIvA cells treated with OHT to induce DSBs, demonstrated by increased γH2AX, and IAA to induce degradation of the AsiSI fusion protein, molecular weight in kDa marked. C qPCR quantification of DSB induction at 3 different loci amplified in iMUT-seq; one NHEJ-prone on chromosome 9, one HR-prone on chromosome 17 and one uncut control on chromosome 1, points represent each biological replicate and error bars are S.D., n = 3 independent biological replicates. D iMUT-seq pipeline. First, genomic DNA from (A) is amplified in a PCR reaction with a multiplex of primers targeting the AsiSI induced DSB sites. This amplifies both DSBs ligated in cis as well as translocated DSBs. This amplified DNA can then be sequenced via NGS, allowing us to profile mutations around DSBs at single-nucleotide resolution, as well as map translocations across the genome. Source data are provided with this paper.

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