GSM5220967: NSD2_NSD2KD_CUT-RUN; Mus musculus; OTHER
This assay was performed as described previously with some modifications. Buffer exchange was performed by centrifugation at 4 °C, 600g for 5 minutes. After the addition of concanavalin A beads, the buffer exchange was performed with a magnetic stand, allowing at least 5 minutes for the beads to completely bind to the magnet. Cells were harvested into a single cell suspension, counted, washed once with ice-cold PBS (Corning) and resuspended with ice-cold Nuclear Extraction (NE) buffer to a final concentration of 10x106 cells/mL. After 10 minutes on ice, cells were washed with Wash Buffer (WB) and resuspended in WB at a final concentration of 5x106 cells/mL. Efficient nuclear extraction was confirmed by Trypan Blue staining. In case of presence of nuclear aggregates, suspension was filtered through a 40um strainer before proceeding to next step. Concanavalin A beads were equilibrated in ice-cold binding buffer and mixed with the isolated nuclei (10ul bead slurry per 5x105 cells). The nuclei-beads mixture was incubated for 10 minutes rotating at 4°C. Successful nuclei-bead binding was confirmed with Trypan Blue staining. Beads-coated nuclei were resuspended in WB at a final concentration of 5x106 cells/mL and 100 μL were aliquoted into each sample tube. While gently vortexing, 100 μL of 2x primary antibody mix was added to the corresponding sample tube. Tube samples were mixed and incubated in rotation overnight at 4°C. Next day, nuclei were washed twice with 1 mL of ice-cold WB and resuspended in 25 μL of ice-cold WB. While gently vortexing, 25 μL of 2x pAG-MNase mix was added to each tube and samples were incubated for 1 hour at 4°C. Nuclei were washed twice with WB and resuspended in 150 μL of ice-cold WB. While gently vortexing, 3 μL of pre-chilled 100 μM CaCl2 were added and after briefly flicking to mix, tubes were incubated for 30 minutes in a 0 °C water bath. After 30 minutes, the MNase was inactivated by adding 150 μL of 2x STOP buffer. To isolate the digested DNA though diffusion, samples were incubated 20 minutes at 37 °C with no shaking. Samples were centrifuged at 16,000g for 5 minutes at 4°C and the resulting supernatant was treated with 3 μL of 10% SDS and 2.5 μL of 20 mg/mL proteinase K at 65 °C for 1 hour to overnight without shaking. DNA was purified with Qiagen MinElute PCR purification kit. Sequencing libraries were constructed using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB E7645L) following the manufacturer's manual.