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==Protocol== *Retrieve cells cultured in T75 flasks. Each vial frozen down should contain ~1-2M cells in 1 mL of media+5%DMSO *Wash cells in PBS by dripping slowly 1 mL of cells from the vial in a 15 mL tube containing 9 mL of PBS. The cells visibly settle to the bottom. *Centrifuge at 300g (rcf) to pellet the cells and remove the supernatant *Make a 20 mL stock of NIB 50 uL of 4M TrisHCl 40 uL of 5M NaCl 60 uL of 1M MgCl2 (950 mg into 10 mL of water) 20 uL of Igepal 2 mL of 1% BSA Protease inhibitor tablets x2 18 mL of water Add everything together in a 50 mL tube and place on rocker for 5 minutes for everything to dissolve. '''Do not agitate as the Igepal and BSA bubbles''' *Resuspend cells in 200uL of NIB+LIS solution for 5 minutes: 195uL NIB+5uL of 500mM lithium 3,5-diiodosalicylic acid (20mg dissolved in 100 mL of water) *The depletion reaction is quenched with the addition of 800uL of NIB. The nuclei can now be added to ligation/enzymatic reactions. *To spin down depleted nuclei, spin at 600g for 15 minutes.
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