Jul 05, 2024
- 1Northwestern
External link: https://doi.org/10.7554/eLife.101035
Protocol Citation: amanda schneeweis 2024. Nuclei Isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6wryl5d/v1
Manuscript citation:
Gaertner Z, Oram C, Schneeweis A, Schonfeld E, Bolduc C, Chen C, Dombeck D, Parisiadou L, Poulin J, Awatramani R Molecular and spatial transcriptomic classification of midbrain dopamine neurons and their alterations in a LRRK2G2019S model of Parkinson’s disease. eLife 13(). doi: 10.7554/eLife.101035
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 05, 2024
Last Modified: July 05, 2024
Protocol Integer ID: 102927
Keywords: nuclei isolation nuclei isolation for snrna, nuclei isolation, snrna, seq
Funders Acknowledgements:
ASAP
Abstract
nuclei isolation for snRNA-seq
Prepare buffers and filter sterilize, add RNAse inhibitor fresh NP40 Lysis Buffer (NST): 0.1% NP-40 Alternative (or NP-40), 10 millimolar (mM) Tris, 146 millimolar (mM) NaCl, 1 millimolar (mM) CaCl2, 21mM MgCl2, 40U/mL of Protector RNAse inhibitor (add fresh day of) ST Wash Buffer: (10mM Tris, 146 Mass Percent NaCl, 1 millimolar (mM) CaCl2, 21mM MgCl2), 0.01% BSA, 40U/mL of Protector RNAse inhibitor (add fresh day of) ST Staining buffer (ST-SB): 2%BSA, 0.02%Tween-20, 10 millimolar (mM) Tris, 146 millimolar (mM) NaCl, 1mM CaCl2, 21mM MgCl2), 40U/mL of Protector RNAse inhibitor (add fresh day of) Note: Keep tissues/homogenate and buffers on ice throughout the protocol. Pre-cool the centrifuge to 4 °C and keep at 4 °C for all steps.
Tissue collection a) Sacrifice and rapidly decapitate mice. Using chilled brain matrix (Ted Pella), cut a thick coronal section from 5mm back from start of cortex until start of cerebellum. Store section in ice-cold Hibernate-A media in 10cm plate on ice until all brains have been dissected. b) Use a razor blade to dissect out midbrain and collect tissue, placing directly into dounce homogenizer.
Tissue lysis and homogenizing a) For each sample to barcode and pool: prepare a separate homogenizer and douncing pestles (loose and tight). Add 1 mL NST buffer to the tube with tissue and transfer to dounce homogenizer and keep on ice. b) with a total volume of 1 mL , dounce 20 times with the loose pestle followed by 20 times with the tight pestle. c) Add 1 mL of ST wash buffer, filter through 30µm filters (Milentyi Biotec 130-041-407) and transfer filtered homogenate to a 15mL tube. d) Rinse the homogenizer with 3x 1 mL of ST wash buffer, filter through 30µm filters and add to the filtered homogenate to add up to a final volume of 5 mL . e) Immediately spin down at 500 g for 00:05:00 at 4 °C to pellet the nuclei in swing bucket rotor f) Remove supernatant g) Resuspend nuclei in 200µl of ST-SB and transfer to lo-bind 1.5 mL tube and wash out the original tube with an additional 1 mL and transfer to the same 1.5ml tube. h) Wash by spinning down for 00:05:00 at 500 g at 4 °C and resuspending in 1.2 mL ST-SB twice, for a total of 3 washes i) resuspend in 1 mL ST-SB
10m
Proceed with sorting nuclei for GFP+ nuclei via MACS Tyto