Nuclei Isolation for Downstream Genomics Applications

Nuclear Isolation

Estimated time

1 - 1.5 hrs depending on the number of samples

  1. Prior to starting, make the Lysis Buffer and Wash Buffer and keep them on ice. Be sure to use freshly made Lysis Buffer and Wash Buffer each time – see recipe at the bottom.

  2. Obtain 50,000 cells for MEFs by trypsin and count accordingly.

    • Optional: if using a spike-in: Add 1000 cells from HEK293Ts to each MEF tube.

  3. Pellet at 500xg for 5 mins at 4C. Aspirate supernatant gently using P1000.

  4. Resuspend the cell pellet in 50 µL of Lysis Buffer by pipetting up and down three times.

  5. Incubate on ice for 3 min. If lysing multiple samples, make sure that all samples are lysed for the same total amount of time by proceeding to Step 7 after 3 min.

  6. Add 1 mL of Wash Buffer to dilute the lysis reagents. Invert the tube five times to mix.

  7. Pellet nuclei at 500g for 10 min at 4°C in a fixed-angle microcentrifuge. Orient the tubes in a consistent fashion so that the pellet will end up in the same location.

  8. STOPPING POINT: Pelleted nuclei can be flash frozen, fixed or immediately processed for downstream genomic applications like ATAC-seq, ChIP-seq, or Chromatin conformation capture.

Recipes

Stock solutions

Make sure you have enough of the following stock solutions. These are often shared with other recipes.

1M Tris pH 7.5 stock solution:

Component

Concentration

g / L

Amount / 10 mL

Tris-HCl

1 M

157.64

1.57g

Elga water

to 9 mL

NaOH

to pH 7.5

~140 uL of 12 N NaOH

Elga water

to 10 mL

5M NaCl stock solution:

Component

Concentration

g / L

Amount / 10 mL

NaCl

5 M

292.21

2.92g

Elga water

to 10 mL

1M magnesium chloride stock solution:

Component

Concentration

g / L

Amount / 10 mL

MgCl2

1 M

95.21

0.952 g

Elga water

to 10 mL

Resuspension Buffer (RSB)- 100mL, scale as needed

Component

Final Concentration

Amount / 100 mL

1 M Tris, pH 7.5

10 mM

1 mL

5 M NaCl

10 mM

200 μL

1M MgCl2

3 mM

300 μL

Elga water

98.5 mL

Cell Lysis Buffer per 50 uL, scale as needed

Component

Volume

Cold RSB (recipe above)

48.5uL

10% (wt/vol) IGEPAL-CA360

0.5uL

10% (wt/vol) Tween-20

0.5uL

1% (wt/vol) digitonin

0.5uL

Total volume

50uL

Wash Buffer per 1000 uL, scale as needed

Stock solutions

Volume

Cold RSB (recipe above)

990uL

10% (wt/vol) Tween-20

10uL