Huy:LabNotes/LAND: Difference between revisions
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*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. | *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 | *Centrifuge at 300g (rcf) to pellet the cells and remove the supernatant | ||
*Resuspend in 5 mL of Nuclei Isolation Buffer (NIB): | *Resuspend in 5 mL of Nuclei Isolation Buffer (NIB) for 20 minutes minimum with gentle mixing on ice: | ||
**Make a 20 mL stock of NIB | **Make a 20 mL stock of NIB | ||
50 uL of 4M TrisHCl | 50 uL of 4M TrisHCl | ||
Line 17: | Line 17: | ||
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''' | |||
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 them in pre-coated 1% BSA tubes | ||
*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 | |||
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Revision as of 05:18, 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) for 20 minutes minimum with gentle mixing on ice:
- 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 them in pre-coated 1% BSA tubes
- 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
==