Nuclei Isolation for Downstream Genomics Applications¶
Nuclear Isolation¶
Estimated time
1 - 1.5 hrs depending on the number of samples
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.
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.
Pellet at 500xg for 5 mins at 4C. Aspirate supernatant gently using P1000.
Resuspend the cell pellet in 50 µL of Lysis Buffer by pipetting up and down three times.
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.
Add 1 mL of Wash Buffer to dilute the lysis reagents. Invert the tube five times to mix.
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.
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 |