Sep 10, 2025
DEFND cryopreserved colon tissue
- Kathy Plaisance1,
- Antonina Mikorska1,
- Thierry Voet1
- 1KULeuven Lab of Reproductive Genomics
Protocol Citation: Kathy Plaisance, Antonina Mikorska, Thierry Voet 2025. DEFND cryopreserved colon tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx47wdl8j/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 tested this protocol, data analysis is still ongoing
Created: August 19, 2025
Last Modified: September 10, 2025
Protocol Integer ID: 226580
Keywords: Cryostat, Homogenization, Permeabilization, colon tissue this protocol, colon tissue, description of defnd, defnd
Funders Acknowledgements:
Aligning Science Across Parkinson's: ASAP
Grant ID: ASAP-025179
Abstract
This protocol details the description of DEFND cryopreserved colon tissue.
Materials
| A | B | C | D | |
| Colon lysis buffer | Stock concentration | Final concentration | V (µL) | |
| TrisHCl pH 7,4 | 1000 mM | 10 % | 10.1 | |
| NaCl | 5000 mM | 10 | 2.0 | |
| MgCl2 | 1000 mM | 3 | 3.0 | |
| DTT | 100 mM | 1 | 10.1 | |
| RNase inhibitor RNasin (Promega) | 40 U/µL | 1 | 25.3 | |
| Nuclease-free water | 959.5 | |||
| Total volume | 1010.0 |
| A | B | C | D | |
| Wash buffer Promega | Stock concentration | Final concentration | V (µL) | |
| TrisHCl pH 7,4 | 1000 mM | 10 mM | 22.2 | |
| NaCl | 5000 mM | 10 mM | 4.4 | |
| MgCl2 | 1000 mM | 3 mM | 6.7 | |
| BSA | 10 % | 1 % | 222.0 | |
| Tween-20 | 10 % | 0.1 % | 22.2 | |
| DTT | 100 mM | 1 mM | 22.2 | |
| RNase inhibitor RNasin (Promega) | 40 U/µL | 1 U/µL | 55.5 | |
| Nuclease-free water | 1864.8 | |||
| Total volume | 2220.0 |
| A | B | C | D | |
| DEFND buffer | Stock concentration | Final concentration | V (µL) | |
| TrisHCl pH 7,4 | 1000 mM | 10 mM | 2.2 | |
| NaCl | 5000 mM | 10 mM | 0.4 | |
| MgCl2 | 1000 mM | 3 mM | 0.7 | |
| Igepal (NP40 substitute) | 10 % | 0.1 % | 2.2 | |
| cOmplete protease inhibitor | 25 X | 1 X | 8.8 | |
| Pefabloc | 20 mg/mL | 1 mg/mL | 11.0 | |
| RNase inhibitor Protector (Sigma) | 40 U/µl | 0.5 U/µl | 2.8 | |
| Lithium diiodosalicylate (LIS) | 100 mM | 12.5 mM | 27.5 | |
| Nuclease-free water | 164.5 | |||
| Total volume | 220.0 |
| A | B | C | D | |
| Nuclei isolation buffer (NIB) | Stock concentration | Final concentration | V (µL) | |
| TrisHCl pH 7,4 | 1000 mM | 10 mM | 10.1 | |
| NaCl | 5000 mM | 10 mM | 2.0 | |
| MgCl2 | 1000 mM | 3 mM | 3.0 | |
| Igepal (NP40 substitute) | 10 % | 0.1 % | 10.1 | |
| cOmplete protease inhibitor | 25 X | 1 X | 40.4 | |
| RNasin (Promega) | 20 U/µl | 0.2 U/µl | 10.1 | |
| Nuclease-free water | 934.3 | |||
| Total volume | 1010.0 |
| A | B | C | D | |
| 1X diluted nuclei buffer | Stock concentration | Final concentration | Volume (µL) | |
| 20X nuclei buffer | 20 X | 1 X | 5.0 | |
| DTT | 100 mM | 1 mM | 1.0 | |
| RNase inhibitor Protector (Sigma) | 40 U/µL | 1 U/µL | 2.5 | |
| Nuclease-free water | 91.5 | |||
| Total volume | 100.0 |
| A | B | C | D | |
| MNase reaction buffer | Stock concentration | Final concentration | Volume (µL) | |
| 20X nuclei buffer | 20 X | 1 X | 5.0 | |
| CaCl2 | 1000 mM | 5 mM | 0.5 | |
| DTT | 100 mM | 1 mM | 1.0 | |
| RNase inhibitor Protector (Sigma) | 40 U/µL | 1 U/µL | 2.5 | |
| Nuclease-free water | 91.0 | |||
| Total volume | 100.0 |
| A | B | C | D | |
| MNase 0,2U/µL | Units in powder | Stock concentration | Volume (µL) | |
| Lyophilised MNase in vial | 50 U | 0.2 U/µL | ||
| Nuclease-free water | 250.0 | |||
| Total volume | 250.0 |
| A | B | C | D | |
| Stop solution | Stock concentration | Final concentration | V (µL) | |
| EGTA | 500 mM | 100 mM | 0.6 | |
| Nuclease-free water | 2.4 | |||
| Total volume | 3.0 |
Remark: CaCl2 concentration may not be optimal, 5µL of 0,1M CaCl2 added to 500µL nuclei and 5µL enzyme is recomended. pH may also not be optimal, as it should be between 9 and 10.
Troubleshooting
Before start
Book necessary instruments and pre-chill centrifuge to 4°C.
Sectioning
Cool cryostat to -18 °C (blade and chamber).
Take sample out of -80 °C and place on dry ice.
Make 6-10 sections of 50 µm and place in DNA LoBind tube.
Homogenization
17m
Note
Perform all following steps on wet ice.
Add 500 µL colon lysis buffer and incubate for 00:01:00 .
1m
Pipet up and down 20 times.
Add an additional 500 µL colon lysis buffer.
Incubate On ice for 00:01:00 and pipet up and down 20 times.
1m
Triturate the sample 20 times using a pellet pestle.
Spin for 00:05:00 at 500 x g and 4 °C , remove the supernatant and resuspend in 1 mL wash buffer Promega.
5m
Transfer the sample with wide-bore pipette onto 40 µm strainer (pluriStrainer mini) placed on 2 mL DNA LoBind tube.
Wash the 1.5 mL tube with additional 500 µL wash buffer Promega, transfer onto the filter.
Spin the 2 mL tube for 00:05:00 at 500 x g and 4 °C .
5m
Remove the supernatant after the centrifugation.
Resuspend the pellet in 700 µL of wash buffer Promega.
Filtrate through flowmi and move to a 20 µm filter (pluriStrainer mini) placed on 2 mL DNA LoBind tube.
Centrifuge 00:05:00 at 500 x g and 4 °C .
5m
Remove the supernatant after the centrifugation.
Permeabilization
10m
Resuspend in 200 µL 12.5 mM DEFND buffer (start incubation time when resuspending).
Incubate On ice for 00:05:00 .
5m
Immediately following this incubation, add 1 mL of NIB and centrifuge the nuclei at 4 °C for 00:05:00 at 500 x g .
5m
Resuspend the pellet in either 25 µL 1X diluted nuclei buffer or 50 µL MNase reaction buffer (CaCl2), depending on choice in step 25 (full Multiome experiment or nucleosome depletion MNase QC).
Dilute further if clumping is present and/or the concentration is too high.
Count nuclei using Propidium Iodide and Acridine Orange (AO/PI).
Nuclei count
Total cell concentration (cells and nuclei):
Live cell concentration (cells):
Dead cell concentration (nuclei):
Viability:
Either continue with the full 10X Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (CG000338 Rev F) protocol without alterations or perform a nucleosome depletion quality control using MNase (Merck), as instructed below.
Note
Adapted from Olsen TR, Talla P, Sagatelian RK, Furnari J, Bruce JN, Canoll P, Zha S, Sims PA. Scalable co-sequencing of RNA and DNA from individual nuclei. Nat Methods. 2025 Mar;22(3):477-487. doi: 10.1038/s41592-024-02579-x. Epub 2025 Feb 12. PMID: 39939719; PMCID: PMC12272635.
Digestion
2m
Transfer 50 µL (or other volume) of nuclei resuspend in reaction buffer to different eppendorf tubes, so split conditions from nucleosome depletion into two.
Preheat ThermoShaker to 28 °C .
Add desired volume of micrococcal nuclease solution (1 unit per 5 µL ) to obtain 7 U of enzyme per 1x106 cells.
Mix gently and constantly for 00:02:00 in a 28 °C ThermoShaker.
2m
At the end of 2 minutes, add 1 µL (or other volume equal to 1/50 from sample volume) of 0.1 Molarity (M) EGTA to stop.
Negative control reaction buffer and reaction buffer + stop solution without MNase.
DNA extraction
Add 100 µL RLT+ buffer to lyse nuclei and vortex.
Extract DNA using DNA Clean & Concentrator®
Protocol references
Olsen TR, Talla P, Sagatelian RK, Furnari J, Bruce JN, Canoll P, Zha S, Sims PA. Scalable co-sequencing of RNA and DNA from individual nuclei. Nat Methods. 2025 Mar;22(3):477-487. doi: 10.1038/s41592-024-02579-x. Epub 2025 Feb 12. PMID: 39939719; PMCID: PMC12272635.