Sep 24, 2024
Nuclei isolation from human brain cortex
- Xian Adiconis1,
- Joshua Levin1
- 1Broad Institute of MIT and Harvard
Protocol Citation: Xian Adiconis, Joshua Levin 2024. Nuclei isolation from human brain cortex. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l626bzgqe/v1
Manuscript citation:
Simmons SK, Adiconis X, Haywood N, Parker J, Lin Z, Liao Z, Tuncali I, Al'Khafaji AM, Shin A, Jagadeesh K, Gosik K, Gatzen M, Smith JT, El Kodsi DN, Kuras Y, Baecher-Allan C, Serrano GE, Beach TG, Garimella K, Rozenblatt-Rosen O, Regev A, Dong X, Scherzer CR, Levin JZ. Experimental and Computational Methods for Allelic Imbalance Analysis from Single-Nucleus RNA-seq Data. bioRxiv [Preprint]. 2024 Aug 16:2024.08.13.607784. doi: 10.1101/2024.08.13.607784. PMID: 39185246; PMCID: PMC11343128.
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: September 18, 2024
Last Modified: September 24, 2024
Protocol Integer ID: 107800
Keywords: nuclei isolation, single-nucleus RNA-seq, preserved brain cortex, human brain cortex this protocol, recombinant rnase inhibitor into the ez, nuclei suspension, human brain cortex, rnalater, brain cortex, adding recombinant rnase inhibitor, incubation step, prep lysis buffer in the incubation step, cell strainer after the tissue, isolation
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000301
Abstract
This protocol can be used on flash-frozen or RNAlater-preserved brain cortex. It is based on a previously published method (1) with a few modifications including, 1) Adding Recombinant RNase Inhibitor into the EZ prep lysis buffer in the incubation steps. 2) Passing the lysate through a 40 µm cell strainer after the tissue grinding. 3) Doing an additional wash. 4) Passing the nuclei suspension through a 20 µm filter before the final counting.
Materials
Reagents and devices
| A | B | C | |
| Item | Vendor | Part Number | |
| Nuclei Isolation Kit: Nuclei EZ Prep | Sigma | NUC101-1KT | |
| Recombinant RNase Inhibitor, 5,000 U, 40 U/µl | Takara | 2313A | |
| 10X Phosphate-Buffered Saline (PBS), molecular biology grade, 1L | Invitrogen | AM9625 | |
| Bovine Serum Albumin (BSA), suitable for molecular biology, 100 mg | Sigma | B6917-100MG | |
| Cellometer ViaStain AO Staining Solution | Nexcelom Bioscience | CS1-0108-5ML | |
| Counting Chamber SD100, 2 counts/slide (75/BX) | Nexcelom Bioscience | CHT4-SD100-002 | |
| KIMBLE Dounce tissue grinder set, 2 mL complete | Sigma | D8938 | |
| 40 µm cell strainer | VWR | 21008-949 | |
| 20 µm sterile single-pack CellTrics filters | Sysmex | 04-004-2325 |
Troubleshooting
Safety warnings
For hazard information and safety warnings, please refer to the MSDSs (Material Safety Data Sheets).
Prepare the following lysis buffers, wash buffer and resuspension buffer, and keep on ice.
Lysis buffer1
| A | B | |
| 1x | ||
| Nuclei EZ lysis buffer | 4 ml | |
| RNase Inhibitor | 20 µl |
Lysis buffer2
| A | B | |
| 1x | ||
| Nuclei EZ lysis buffer | 4 ml | |
| RNase Inhibitor | 4 µl |
Wash buffer (PBS with 0.01% BSA and 0.04 U/µl RNase Inhibitor)
| A | B | |
| 1x (ml) | ||
| 10x PBS | 1 | |
| 2% BSA | 0.05 | |
| RNase Inhibitor | 0.01 | |
| H2O | 8.94 | |
| Total | 10 |
Nuclei resuspension buffer (PBS with 1% BSA and 0.2 U/µl RNase Inhibitor)
| A | B | |
| 1x (ml) | ||
| 10x PBS | 0.1 | |
| 2% BSA | 0.5 | |
| RNase Inhibitor | 0.005 | |
| H2O | 0.395 | |
| Total | 1 |
15m
Add 2 mL of cold Lysis buffer1 into an empty douncing tube and keep on ice.
Drop the frozen tissue into the douncing tube and submerge it in the lysis buffer. Start immediately grinding the tissue with pestle A 25 times or until resistance disappears. Continue grinding with pestle B 25 times. Pass the lysate through a 40 µm filter into a new 50 mL tube on ice.
15m
Rinse the douncing tube with the remaining 2 mL Lysis buffer1, pass through the same filter and into the same 50 mL tube.
Mix the lysate well by inverting the tube 5 times and incubate on ice for 00:05:00 .
5m
Pellet the nuclei by 500 x g, 4°C, 00:05:00 . Carefully aspirate the supernatant and set the nuclei pellet on ice.
5m
Resuspend the nuclei by adding 1 mL Lysis buffer2 and mix well with a P1000 pipette. Add the remaining 3 mL Lysis buffer2, mix well by inverting, and incubate on ice for 00:05:00
5m
Pellet the nuclei by 500 x g, 4°C, 00:05:00 as in step 6. Carefully aspirate the supernatant and set the nuclei pellet on ice.
5m
Resuspend the pellet with 750 µL Nuclei resuspension buffer, filter through a 20 µm filter, and collect into a 1.5 ml Eppendorf tube. Count the nuclei using Cellometer K2 (Nexcelom Bioscience) with the AO stain. Keep on ice until downstream processing.
Protocol references
1. Habib N, Li Y, Heidenreich M, Swiech L, Avraham-Davidi I, Trombetta JJ, et al. Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons. Science. 2016;353(6302):925-8.