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*Pelleted nuclei are then resuspended in 200uL of NIB+LIS solution for 5 minutes:
*Pelleted nuclei are then resuspended 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)
  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
*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 6000g for 15 minutes.
 
 
 
==Microscope Pictures==
==Microscope Pictures==
*Insert pictures of depleted and isolated nuclei here
*Insert pictures of depleted and isolated nuclei here
==Important Notes==
==Important Notes==
*The addition of 4X the LIS amount during depletion results in a 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 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
*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
  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.
*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.

Revision as of 17:54, 10 May 2017

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
  • Resuspend in 5 mL of Nuclei Isolation Buffer (NIB) for 20 minutes minimum with gentle mixing on ice and transfer to a 5mL tube pre-coated with 1% BSA:
    • 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
  • Isolated nuclei are then centrifuged at 800g (rcf) for 3 minutes to pellet and the supernatant is removed
  • Pelleted nuclei are then resuspended 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 6000g 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.