Nuclei Isolation for Downstream Genomics Applications¶
Nuclear Isolation¶
Expected time: 1 - 1.5 hrs depending on the # 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.
Obtain 50,000 cells for MEFs by trypsin and count accordingly. Optional if using a Spike: Add 1000 cells from HEK293Ts.
If using spike, add appropriate cell volume of Spike cells (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¶
Resuspension Buffer (RSB)- 100mL, scale as needed
Stock solutions |
Working stock |
|---|---|
1M Tris-HCl pH 7.50 - 1mL |
10mM Tris-HCl pH 7.50 |
5M NaCl - 200uL |
10mM NaCl |
1M MgCl2 - 300uL |
3mM MgCl2 |
UltraPure distilled H2O - 98.5mL |
Cell Lysis Buffer per 50 uL, scale as needed
Stock solutions |
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 |