GSM6361078: CUT&RUN_MED1_mESC_NSD1-dTAG_dTAG-13_1h; Mus musculus; OTHER
Dessociated cells were bound to Concanavalin A beads and permeabilized with Digitonin, followed by binding of antibodies and pAG-MNase to targets on native chromatin in situ. MNase cleavage of target occupied DNA was activated by addition of CaCl2 at 4C. Cleaved DNA was released by incubation at 37C and purified using Monarch DNA Cleanup Kit (NEB). Libraries were prepared with less than 10 ng CUT&RUN DNA using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) according to manufacturer's protocol with modifications: end prep was performed for 30 min at 20C followed by 1 h at 50C to prevent melting of small fragments, adaptor-ligated DNA was cleaned up without size selection, and the anneal/extension step of PCR amplification was performed for 10s at 65C to favor exponential amplification of the desired CUT&RUN fragments.