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(Created page with "=Lithium Assisted Nucleosome Depletion= ==Background== Previously published under "Sequencing thousands of single-cell genomes with combinatorial indexing" by Andrew Adey ==Ai...")
 
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  Protease inhibitor tablets x2
  Protease inhibitor tablets x2
  18 mL of water
  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'

Revision as of 02:51, 9 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):
    • 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'