GSM9741529: E11.5 AGM CD31 EC CUT&RUN, Input; Mus musculus; OTHER
CUT&RUN was performed on ECs using the CUT&RUN Assay Kit (Cell Signaling Technology, Cat. No. 86652) per the manufacturer's instructions. Briefly, ECs were harvested, washed, and bound to concanavalin A-coated magnetic beads. Bead-bound cells were permeabilized and incubated with primary antibody at 4 °C overnight with gentle agitation. After washing, cells were incubated with recombinant pAG-MNase for 1 hour at 4 °C. Targeted cleavage was initiated by adding calcium chloride and carried out for 30 minutes at 4 °C. Reactions were stopped with stop buffer, and released chromatin fragments were collected from the supernatant. An input control (antibody untreated) was processed in parallel to assess background and normalize enrichment. DNA was purified using kit spin columns, and libraries were prepared with the NEBNext DNA Library Prep Kit for Illumina (New England Biolabs, Cat. No. E7645).