TRIP assay of mismatch repair during single-strand annealing

Biases of DNA repair can shape the nucleotide landscape of genomes at evolutionary timescales. However, such biases have not yet been measured in chromatin for lack of technologies. Here we develop a genome-wide assay whereby the same DNA lesion is repaired in different chromatin contexts. We insert thousands of barcoded transposons carrying a reporter of DNA mismatch repair in the genome of mouse embryonic stem cells. Upon inducing a double-strand break between tandem repeats, a mismatch is generated when the single strand annealing repair pathway is used. Surprisingly, the mismatch repair machinery favors the same strand 60-80% of the time. The location of the lesion in the genome and the type of mismatch have little influence on the repair bias in this context. Five constructs were used, each producing a different mismatch upon induction of a double-strand break (A:G, T:G, A:C, T:C and C:A, where the positions of the nucleotides are on opposite strands in A:C and C:A). Each construct was used to set two independent TRIP pools in mouse E14 ES cells. For each TRIP pool, two mapping experiments by inverse PCR (iPCR) were performed in three total batches. Then for each TRIP pool, two independent I-SceI transfections were performed and genomic DNA was collected before the transfection (no I-SceI control), or 24 hour and 48 hour after transfection. The state of the DNA after repair was assayed by either UMI-LA (linear amplification) or UMI-PCR. Each method was used twice on a given genomic DNA extraction.