Nuclei isolation from frozen tissue
Emily Stephenson, Elena Prigmore, agnes oszlanczi, di zhou
Disclaimer
Number of homogenisation strokes will be tissue specific as well as cell lysis, this may need to be optimised per tissue.
Abstract
This protocol describes a method for the isolation of intact nuclei for further downstream methods such as single nuclei RNA-seq, ATAC-seq or 10x Multiome.
The protocol is adapted from Nadelmann et al doi: 10.1002/cpz1.132
Before start
Tissue can be collected before starting and kept on dry ice. Frozen tissue as small as a grain of rice can be used or OCT-embedded tissue sections.
Steps
Prepare reagents and buffers
Prepare nuclei isolation buffer 1 (NIM1) according to the table below. NIM1 can be made, filtered and stored at 4°C for 6 months. Record date on top of tube.
| A | B | C |
|---|---|---|
| Sucrose 342.3 g/mol | 4.279g | 250 |
| 2M KCL | 625ul | 25 |
| 1M MgCl2 | 250ul | 5 |
| 1MM Tris buffer | 500ul | 10 |
| Nuclease free water | 48.625ml | - |
| Total | 50ml |
Prepare nuclei isolation buffer 2 (NIM2) according to the table below. NIM2 must be made on the day of experiment and kept on ice. Approx. 5mL per sample will be needed.
- Pre dilute 1M DTT in 4.995ml water
- For 100X protease inhibitor stock, dissolve 1 tablet in 500ul nuclease free water. Stock solution can be kept at 4°C for 2 weeks or 12 weeks at -20°C (record date on top of tube).
| A | B | C |
|---|---|---|
| NIM1 | 4946ul | 1x |
| 1mM DTT | 5ul | 1uM |
| 100x protease inhibitor | 50ul | 1x |
| Total | 5mL |
Prepare homogenisation buffer (HB) according to the table below. HB must be made on the day of experiment and kept on ice. Do not vortex. Approx. 5mL per sample will be needed.
| A | B | C |
|---|---|---|
| NIM2 | 4850ul | 1x |
| RNasin plus 40 U/ul | 50ul | 0.4U.ul |
| Superasin 20 U/ul | 50ul | 0.2U/ul |
| Triton X-100 10% | 50ul | 0.1% |
| Total | 5ml |
Prepare wash buffer (WB) according to the table below. WB must be made on the day of experiment and kept on ice. Do not vortex. Make approx. 1ml per sample, more may be needed if performing extra washes or clean-up steps.
| A | B | C |
|---|---|---|
| 1x PBS | 975ul | 1x |
| BSA powder | 0.05g | 5% |
| Rnasin protector | 25ul | 40U/ul |
| Total | 1ml |
Optional step: make up lymphoprep solution according to table below if samples have a lot of debris that need to be removed. Keep on ice.
| A | B | C |
|---|---|---|
| Lymphoprep | 1350ul | 90% |
| 10x PBS | 150ul | 1x |
| Total | 1500ul |
Nuclei isolation
Collect tissue or tissue sections on dry ice and transfer to the dounce homogeniser
Add 3mL homogenisation buffer to the tissue.
If using tissue embedded in OCT, leave for 5 mins on ice. Mix half way and use pestle A to push OCT to the bottom of the homogeniser. If OCT still remains, keep mixing until dissolved.
On ice, gently homogenise the tissue using pestle A. Up to 20 strokes may be needed until no more resistance is felt.
Rinse pestle with 500mL homogenisation buffer.
On ice, gently homogenise the tissue using pestle B. Up to 20 strokes may be needed until no more resistance is felt.
Rinse pestle with 500mL homogenisation buffer.
Pour homogenate through a 40um cell strainer into a 50mL Falcon tube on ice. Rinse homogeniser with 500mL homogenisation buffer. Transfer any droplets underneath the filter with a pipette.
Centrifuge the tube at 500 x g for 6 mins at 4°C. Acc 0, dec 3.
Carefully remove the supernatant with 1mL pipette and transfer to another tube, do not throw away.
Add 500mL wash buffer and leave for 2 mins on ice.
Resuspend nuclei with a wide bore tip and transfer to 1.5mL eppendorf tube.
Look at nuclei underneath microscope using filtered trypan blue and C-Chip. If there appears to be a lot of debris, either pass nuclei through a 40mM FlowMi filter or perform a clean-up (steps at the end of protocol), if not proceed to count.
Nuclei will stain blue with trypan. Count nuclei in all four corners then use the following calculation: (count/4) x 2 (trypan dilution factor) x 10 (volume factor) = conc. (nuclei per mL)
Multiply by 500 to get total nuclei. Record this number as the “unclean count”.
Centrifuge the tube at 500 x g for 3 mins at 4°C.
If unclean count is more than 500k nuclei, remove as much supernatant as possible without disturbing pellet and wash again with 500ml of wash buffer. If count is less than 500k, remove supernatant and leave 10-50mL in tube depending on count (use tube of water as a guide). Do not throw away supernatant.
Resuspend pellet gently and count nuclei using filtered trypan blue and C-Chip. May need to do a 1 in 10 dilution using the supernatant as diluent. Count nuclei in all four corners then use the following calculation:
(count/4) x 2 (trypan dilution factor) x 10 (volume factor) x 10 (dilution factor) = conc. (nuclei per mL)
Multiply by volume to get total nuclei. Record this number as the “clean count”.
Proceed with next protocol such as 10x Genomics Multiome or scRNA-seq.
Optional clean-up to remove debris
Add 475ml wash buffer to 2 Eppendorf tubes (not LoBind) for each sample.
Add 22ul nuclei solution followed by 300ul 90% lymphoprep to each tube. Mix by inverting (do not pipette mix).
Centrifuge sample at 20,000 x g for 15mins at 4°C.
Remove top 500-800mL layer and add 500mL wash buffer. Be careful not to disrupt the nuclei ‘cloud’ which will hopefully be visible near the bottom of the tube, but may not be seen as a pellet. Mix by pipetting using a wide bore tip.
Proceed to step 19 in original protocol.
