Huy:LabNotes/LAND: Difference between revisions
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>HuyLam (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'