Fig. 1: UMI-DSBseq quantitative single-molecule sequencing of DSBs and repair products at three targets in tomato.
From: Uncovering the dynamics of precise repair at CRISPR/Cas9-induced double-strand breaks
A Collection of time-course: mesophyll cell protoplasts are isolated from 2 to 3 week-old seedlings of M82 Solanum lycopersicum. Duplicate samples are prepared with 200,000 protoplasts for each of the 7 time-points along 72 h. CRISPR RNPs are introduced by PEG-mediated transformation. Samples are frozen at 0, 6, 12, 24, 36, 48, and 72 h after RNP introduction and DNA is extracted. B UMI-DSBseq target design: a primer specific to the target sequence (blue arrow), is coupled with a restriction enzyme site flanking the sgRNA target sequence to create an available end on intact molecules (WT or Indel) for ligation of adaptors. C UMI-DSBseq library preparation: DNA extraction from time-course collection, containing WT (1), unrepaired DSBs (2), and intact molecules containing indels (3), is restricted in vitro with the restriction enzyme identified flanking the target cut site (dashed oval). Following end-repair by fill-in and A-addition, Y-shaped adaptors composed of P7 Illumina flow-cell sequences and containing i7 indexes (orange) and 9 bp unique molecular identifiers (UMIs) in purple, are ligated to the unrepaired DSBs and restricted ends. Target-specific amplification by ligation-mediated PCR follows, with one primer identical to the adaptor sequence and containing the P7 Illumina tail (orange arrow) and one primer specific to the target sequence (blue arrow) with the P5 Illumina tail (red). This results in the amplification of a single end of the DSB between the SpCas9 cut site and the primer. The red X represents the non-captured end of the DSB.
