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=Lithium Assisted Nucleosome Depletion= ==Background== Previously published under "Sequencing thousands of single-cell genomes with combinatorial indexing" by Andrew Adey ==Aim== To isolate nuclei from cells and "deplete" them of the histones so that the DNA is accessible to enzymatic reactions and adapter ligation. ==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. ==Microscope Pictures== *Insert pictures of depleted and isolated nuclei here ==Important Notes== *The addition of 4X the LIS amount during depletion results in an extremely viscous solution. It is unclear whether the LIS lyses nuclei. This viscous solution prevents the centrifugation of nuclei and the LIS reagent couldn't be removed *The depleted nuclei may need to be centrifuged at a much higher speed than undepleted nuclei based on https://www.ncbi.nlm.nih.gov/pmc/articles/PMC148640/pdf/273779.pdf Depleted nuclei were centrifuged at 6000g for 15-20 minutes *The paper also suggests that the LIS "extracts" the histone binding proteins from the nuclei causing a DNA halo of DNA strands that permeate out of the nuclei, but do not actually rupture the nuclei. The picture in the publication needs to be inserted here.
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