Jun 27, 2023

Nuclei isolation from frozen tissue

DOI
https://dx.doi.org/10.17504/protocols.io.8epv5x6wjg1b/v1
Nuclei isolation from frozen tissue
  • 1Newcastle University;
  • 2Wellcome Sanger Institute
  • Human Cell Atlas Method Development Community
Emily Stephenson
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.8epv5x6wjg1b/v1
Protocol Citation: Emily Stephenson, Elena Prigmore, agnes oszlanczi, di zhou 2023. Nuclei isolation from frozen tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x6wjg1b/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 27, 2023
Last Modified: June 27, 2023
Protocol  Integer ID: 84077
Keywords: nuclei isolation from frozen tissue, downstream methods such as single nuclei rna, single nuclei rna, nuclei isolation, intact nuclei, rna, nuclei, method for the isolation, frozen tissue, 10x multiome, isolation
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


Materials
2M KCL
1M MgCL2
1M Tris buffer
Nuclease free water
1mM DTT
100x protease inhibitor
RNasin plus 40 U/ul
Superasin 20 U/ul
Triton X-100 10%
1x PBS
BSA powder
RNasin protector
Lymphoprep
10x PBS
Dounce homogeniser with pestle A and B
40uM cell strainer
Wide bore pipette tips
Trypan blue
Eppendorf tubes
C-chip haemocytometer



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.
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.


ReagentVolume/amountFinal conc. (mM)
Sucrose 342.3 g/mol4.279g250
2M KCL625ul25
1M MgCl2250ul5
1MM Tris buffer500ul10
Nuclease free water48.625ml-
Total50ml

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).


ReagentVolumeFinal conc.
NIM14946ul1x
1mM DTT5ul1uM
100x protease inhibitor50ul1x
Total5mL

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.


ReagentVolumeFinal conc.
NIM24850ul1x
RNasin plus 40 U/ul50ul0.4U.ul
Superasin 20 U/ul50ul0.2U/ul
Triton X-100 10%50ul0.1%
Total5ml

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.


ReagentVolumeFinal conc.
1x PBS975ul1x
BSA powder0.05g5%
Rnasin protector25ul40U/ul
Total1ml





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.


ReagentVolumeFinal conc.
Lymphoprep1350ul90%
10x PBS150ul1x
Total1500ul

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.
Protocol references