Nuclei extraction protocol for CUTANA™ assays

Important Notes

  • Nuclei Extraction Buffer is prepared fresh on the day of nuclei harvest.

  • The CUTANA™ Nuclei Extraction Protocol is optimized for CUT&RUN and CUT&Tag workflows. For CUT&RUN, start with 500,000 cells per reaction; for CUT&Tag, start with 100,000 cells per reaction. Harvest 10% excess cells to account for sample loss.

  • To create 25X Protease Inhibitor stock solution: dissolve 1 Protease Inhibitor tablet (table above) in 2 mL molecular biology grade water. Leftover 25X Protease Inhibitor stock solution can be stored for 12 weeks at -20˚C.

Materials Needed

Reagent

Source

Pre-Nuclei Extraction Buffer

EpiCypher 21-1026

1M Spermidine

Protease Inhibitor, such as Roche cOmplete™, EDTA-free Protease Inhibitor Cocktail

Roche 11873580001

Phosphate Buffered Saline (PBS)

Any vendor

0.4% Trypan Blue

Invitrogen T10282

Brightfield or phase microscope + hemacytometer slides

Any vendor

Nuclei Prep Protocol

  1. Prepare Nuclei Extraction Buffer in a fresh tube. Per reaction, combine 235 µL Pre-Nuclei Extraction Buffer, 0.13 µL Spermidine, and 9.8 µL 25X Protease Inhibitor.

    • If preparing buffer for fewer than 8 reactions, dilute the 1M Spermidine stock 1:10 in molecular biology-grade water and add 1.3 μL per reaction.

  2. Place final Nuclei Extraction Buffer on ice.

  3. Count fresh or frozen cells and confirm starting cell integrity, morphology, and viability by Trypan Blue staining.  It is important that cells have good starting viability. For K652 cells, we aim for >90% viability.

  4. Harvest cells. For CUT&RUN, harvest 500,000 cells per reaction; for CUT&Tag, harvest 100,000 cells per reaction. Harvest 10% excess cells to account for sample loss.

  5. Spin cells at 600 x g for 3 min at room temperature (RT). Remove supernatant and resuspend cells in 100 µL per reaction cold Nuclei Extraction Buffer.

  6. Incubate on ice for 10 min.

  7. Spin at 600 x g for 3 min at 4ºC. Remove and discard supernatant. The pellet should change in appearance from sticky, pale yellow (cells) to white and fluffy (nuclei).

  8. Gently resuspend nuclei in 105 µL per reaction cold Nuclei Extraction Buffer.

  9. Take a 10 µL aliquot to examine nuclear integrity by Trypan Blue staining. Isolated nuclei will stain Trypan Blue positive and should have clear borders and minimal debris. Intact cells will be bright white and round (see Figure 1). For accurate nuclei counts, record "dead" cell numbers on an automated cell counter or manually count blue stained nuclei.

  10. Proceed to desired assay (CUT&RUN or CUT&Tag). If freezing nuclei for later use, see cryopreservation protocol below.


nuclei extraction NEW

Figure 1. Nuclei successfully isolated with CUTANA Nuclei Extraction Protocol. Viable starting cells exclude Trypan Blue (top row), while isolated nuclei stain Trypan Blue positive (bottom row). 

Nuclei Cryopreservation and Thawing Protocol

  1. Aliquot nuclei resuspended in Nuclei Extraction Buffer. EpiCypher scientists typically aliquot for >8 reactions (800 µL + plus 20-30% excess) to account for sample loss at thaw steps.

  2. Slowly freeze aliquots in an isopropanol-filled chiller in a -80°C freezer.

  3. When ready to use samples, thaw nuclei quickly by placing on 37°C block. Move quickly to avoid nuclear lysis and chromatin fragmentation.

  4. Thawed nuclei in Nuclei Extraction Buffer can be directly added to activated ConA beads in both CUT&RUN and CUT&Tag.