TITLE: Nuclei isolation from frozen tissue
AUTHORS: Emily Stephenson, Elena Prigmore, agnes oszlanczi, di zhou

[DESCRIPTION]
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]
SECTION: Prepare reagents and buffers
1. 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.


 

SECTION: Prepare reagents and buffers
2. 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).


 

SECTION: Prepare reagents and buffers
3. 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.


 

SECTION: Prepare reagents and buffers
4. 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.


 





SECTION: Prepare reagents and buffers
5. 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.


 

SECTION: Nuclei isolation
6. Collect tissue or tissue sections on dry ice and transfer to the dounce homogeniser
SECTION: Nuclei isolation
7. Add 3mL homogenisation buffer to the tissue.
SECTION: Nuclei isolation
8. On ice, gently homogenise the tissue using pestle A. Up to 20 strokes may be needed until no more resistance is felt.
SECTION: Nuclei isolation
9. Rinse pestle with 500mL homogenisation buffer.
SECTION: Nuclei isolation
10. On ice, gently homogenise the tissue using pestle B. Up to 20 strokes may be needed until no more resistance is felt.
SECTION: Nuclei isolation
11. Rinse pestle with 500mL homogenisation buffer.
SECTION: Nuclei isolation
12. 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.
SECTION: Nuclei isolation
13. Centrifuge the tube at 500 x g for 6 mins at 4°C. Acc 0, dec 3.
SECTION: Nuclei isolation
14. Carefully remove the supernatant with 1mL pipette and transfer to another tube, do not throw away.
SECTION: Nuclei isolation
15. Add 500mL wash buffer and leave for 2 mins on ice.
SECTION: Nuclei isolation
16. Resuspend nuclei with a wide bore tip and transfer to 1.5mL eppendorf tube.
SECTION: Nuclei isolation
17. 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.
SECTION: Nuclei isolation
18. 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”.
SECTION: Nuclei isolation
19. Centrifuge the tube at 500 x g for 3 mins at 4°C.
SECTION: Nuclei isolation
20. 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.
SECTION: Nuclei isolation
21. 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”.
SECTION: Nuclei isolation
7.1.  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.
SECTION: Nuclei isolation
22. Proceed with next protocol such as 10x Genomics Multiome or scRNA-seq.
SECTION: Optional clean-up to remove debris
23. Add 475ml wash buffer to 2 Eppendorf tubes (not LoBind) for each sample.
SECTION: Optional clean-up to remove debris
24. Add 22ul nuclei solution followed by 300ul 90% lymphoprep to each tube. Mix by inverting (do not pipette mix).
SECTION: Optional clean-up to remove debris
25. Centrifuge sample at 20,000 x g for 15mins at 4°C.
SECTION: Optional clean-up to remove debris
26. 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.
SECTION: Optional clean-up to remove debris
27. Proceed to step 19 in original protocol.