mus81_mec1_sml1_S_Rec_2h
We used i-BLESS (immobilized-BLESS), a new method, particularly applicable to small cells and optimized here for yeast, for direct in situ genome-wide DSB labeling in agarose beads. Cells are embedded in agarose beads using modified Overhausers protocol (what protects DNA from mechanical damage and enables efficient diffusion of the reagents), followed with spheroplasting, lysis and protein removal. Similarly to original BLESS protocol, the dsDNA ends are blunted, 5'-phosphorylated and ligated with biotinylated adapter (proximal). DNA is isolated from agarose, fragmented, and biotinylated fragments are captured on streptavidin beads. The free ends of the captured fragments are then ligated to a second adapter (distal), and the resulting DNA is linearized, amplified by PCR, and sequenced.
SUB6425761
LBSB-17-008