Jul 04, 2023
Nuclei isolation and permeabilisation of fresh frozen human brain samples for 10X Genomics Multiome
This protocol is a draft, published without a DOI.
- Koen Theunis1,
- Suresh Poovathingal2,
- Sarah Geurs3
- 1Laboratory of Computational Biology, VIB-KU Leuven, Center for Brain & Disease Research, Leuven, Belgium;
- 2VIB-KU Leuven/VIB Center for Brain & Disease Research, Leuven, Belgium;
- 3VIB Tech Watch, VIB Technologies, Ghent, Belgium
Protocol Citation: Koen Theunis, Suresh Poovathingal, Sarah Geurs 2023. Nuclei isolation and permeabilisation of fresh frozen human brain samples for 10X Genomics Multiome. protocols.io https://protocols.io/view/nuclei-isolation-and-permeabilisation-of-fresh-fro-cwn7xdhn
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: July 03, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84415
Keywords: ASAPCRN, single nuclei preparation, multiome, snATAC-seq, snRNA-seq, 10X Genomics
Funders Acknowledgement:
ASAP
Abstract
This protocol details the procedure of nuclei isolation and debris removal from fresh frozen brain tissue in preparation for the 10X Genomics multiome ATAC/GEX assay.
Materials
HOW MANY SAMPLES TO PROCESS? 2
| A | B | C | D | E | F | |
| 2X salt-Tris solution | Stock conc | Final conc | V (µl) | |||
| NaCl | 5000 | mM | 292 | mM | 1226,4 | |
| Tris pH7.5 | 1000 | mM | 20 | mM | 420,0 | |
| CaCl | 1000 | mM | 2 | mM | 42,0 | |
| MgCl2 | 1000 | mM | 42 | mM | 882,0 | |
| Water | 18429,6 | |||||
| Total Volume | 21000,0 |
| TST+NP Homogenization buffer | Stock conc | Final conc | V (µl) | |||
| 2X salt-Tris solution | 2 | X | 1 | X | 1100,0 | |
| Tween-20 | 10 | % | 0,03 | % | 6,6 | |
| NP-40 | 10 | % | 0,03 | % | 6,6 | |
| BSA | 7,5 | % | 1 | % | 293,3 | |
| KCl | 3000 | mM | 25 | mM | 18,3 | |
| Sucrose | 2000 | mM | 250 | mM | 275,0 | |
| DTT | 1000 | mM | 1 | mM | 2,2 | |
| cOmplete protease inhibitor | 50 | X | 1 | X | 44,0 | |
| Rnase In plus (promega) | 40 | U/ul | 1 | U/ul | 55,0 | |
| Water | 398,9 | |||||
| Total Volume | 2200,0 |
| Wash buffer 1 | Stock conc | Final conc | V (µl) | |||
| 2X salt-Tris solution | 2 | X | 1 | X | 561,0 | |
| BSA | 7,5 | % | 1 | % | 149,6 | |
| KCl | 3000 | mM | 25 | mM | 9,4 | |
| Sucrose | 2000 | mM | 250 | mM | 140,3 | |
| DTT | 1000 | mM | 1 | mM | 1,1 | |
| cOmplete protease inhibitor | 50 | X | 1 | X | 22,4 | |
| Rnase In plus (promega) | 40 | U/ul | 1 | U/ul | 28,1 | |
| Water | 210,2 | |||||
| Total Volume | 1122,0 |
| GM (Gradient Medium) | Stock conc | Final conc | V(µl) | |||
| CaCl | 1000 | mM | 1 | mM | 1,3 | |
| Optiprep | 60 | % | 50 | % | 1083,3 | |
| MgCl2 | 1000 | mM | 5 | mM | 6,5 | |
| Tris pH 7.5 | 1000 | mM | 10 | mM | 13,0 | |
| Sucrose | 2000 | mM | 75 | mM | 48,8 | |
| DTT | 1000 | mM | 1 | mM | 1,3 | |
| cOmplete protease inhibitor | 50 | X | 0,5 | X | 13,0 | |
| Rnase In plus (promega) | 40 | U/ul | 1 | U/ul | 32,5 | |
| Water | 100,3 | |||||
| Total Volume | 1300 |
| ODM (Optiprep Diluent Medium) | Stock conc | Final conc | V(µl) | |||
| KCl | 3000 | mM | 150 | mM | 62,0 | |
| MgCl2 | 1000 | mM | 30 | mM | 37,2 | |
| Tris pH 8 | 1000 | mM | 60 | mM | 74,4 | |
| Sucrose | 2000 | mM | 250 | mM | 155,0 | |
| RNAse in plus (promega) | 40 | U/uL | 0,5 | U/uL | 15,5 | |
| Water | 895,9 | |||||
| Total Volume | 1240,0 |
| 29% Cushion | Stock conc | Final conc | V(µl) | |||
| Optiprep | 60 | % | 29 | % | 966,7 | |
| ODM | 1033,3 | |||||
| Total Volume | 2000 |
| 1x Lysis | Stock conc | Final conc | V (µl) | |||
| Tris-HCl (pH 7.4) | 1000 | mM | 100 | mM | 50,0 | |
| NaCl | 5000 | mM | 100 | mM | 10,0 | |
| MgCl2 | 1000 | mM | 30 | mM | 15,0 | |
| Tween-20 | 10 | % | 0,1 | % | 5,0 | |
| NP-40 | 10 | % | 0,1 | % | 5,0 | |
| Digitonin | 5 | % | 0,02 | % | 2,0 | |
| Water | 413,0 | |||||
| Total Volume | 500,0 |
| 0.1x Lysis | Stock conc | Final conc | V (µl) | |||
| 1x lysis | 1 | x | 0,1 | x | 100,0 | |
| BSA | 7,5 | % | 1 | % | 133,3 | |
| DTT | 1000 | mM | 1 | mM | 1,0 | |
| Rnase Inhibitor (Promega) | 40 | U/ul | 1 | U/ul | 25,0 | |
| Water | 740,7 | |||||
| Total Volume | 1000,0 |
| Wash Buffer 2 | Stock conc | Final conc | V (µl) | |||
| Tris-HCl (pH 7.4) | 1000 | mM | 10 | mM | 22,0 | |
| NaCl | 5000 | mM | 10 | mM | 4,4 | |
| MgCl2 | 1000 | mM | 3 | mM | 6,6 | |
| BSA | 7,5 | % | 1 | % | 293,3 | |
| Tween-20 | 10 | % | 0,01 | % | 2,2 | |
| DTT | 1000 | mM | 1 | mM | 2,2 | |
| Rnase Inhibitor (SIGMA) | 40 | U/ul | 1 | U/ul | 55,0 | |
| Water | 1814,3 | |||||
| Total Volume | 2200,0 |
| 1X Diluted Nuclei buffer | Stock conc | Final conc | V(µl) | |||
| Nuclei buffer | 20 | X | 1 | X | 7,5 | |
| DTT | 100 | mM | 1 | mM | 1,5 | |
| Rnase Inhibitor (SIGMA) | 40 | U/ul | 1 | U/ul | 3,75 | |
| Water | 137,25 | |||||
| Total Volume | 150 |
Preparation
Preparation
Prepare the 2X salt-Tris solution and aliquot to 1 ml tube and freeze at -20°C
Prepare the BSA in PBS and filter through 0.2um filter.
Prepare the Tween-20 Homogenization lysis buffer fresh for each preperations
Filter sucrose 2M solution through 0.2um/0.45um filter
Prepare cOmplete protease inhibitor (50X) by dissolving a tablet in 1 mL of water.
Filter protease inhibitor solution through 0.2um filter
Place the homogenizer at -80°C at the start and leave 10 min before homgenization on ice.
Homogenization (Peform all steps on ice)
Homogenization (Peform all steps on ice)
Cut brain piece on dry ice (if a large section), then transfer immediately in homogenizer containing 250 uL of TST+NP Homogenization Lysis Buffer. Immediately add 500uL TST+NP Homogenization Lysis Buffer
Allow the tissue to thaw for 2 mins in the homogenization buffer.
Homogenize tissue with pestle A (10X), wait 1min, and with pestle B (10X).
Put the homogenate through 70µm cell strainer and place the falcon on ice. Incubate on ice for 5 mins. Rinse dounce and filter with 250uL TST+NP HB.
During the incubation (in about 4 mins), use LUNA-fl counter to assess the viability of the nuclei.
Transfer the contents from the 50 mL falcon to a 2 mL protein lo-bind tube.
Centrifuge @ 500xg for 5 mins. Discard the supernatant.
Resuspend the pellet in 200 uL of Wash Buffer 1, and transfer to a 2mL DNA lobind tube.
Add additional 320 uL Wash Buffer 1 to a final volume of 520 uL.
Add 520uL of Gradient medium to sample (Vf = 1040 uL).
Isolation by centrifugation
Isolation by centrifugation
Layer 770 uL of 29% Cushion in the ultracentrifuge tube.
Layer the sample on top of cushion using a P1000, without disturbing the cushion.
Optional: Check tube weight, adjust weight to counter differences.
Centrifuge at least 3,000 rcf in a swinging bucket, at 4°C for 20 minutes with brake off.
Remove supernatant, remove the lower supernatant with P200, leaving about 50-100 uL.
Resuspension and permeabilisation
Resuspension and permeabilisation
Gently resuspend nuclei in ultracentrifuge tube and transfer to 1.5 mL DNA lobind tube.
Rinse ultracentrifuge tube with Wash buffer 2 and transfer also to 1.5 mL DNA lobind tube.
Centrifuge @ 350-450xg for 5 mins. Discard the supernatant.
Resuspend in 0.1x lysis buffer (200 uL) and gently pipetmix 5x
Incubate on ice for 2 min
Add Wash buffer 2 (1mL) & gently pipetmix 5x
Centrifuge @ 350-450xg for 5 mins. Discard the supernatant.
Resuspend in 1X Diluted Nuclei buffer. Count