Dec 01, 2025
Version 1
SPLiT-seq Nuclei isolation for Micro-dissected Mouse Brain Tissue V.1
- Ashley Robbins1
- 1University of Pennsylvania
Protocol Citation: Ashley Robbins 2025. SPLiT-seq Nuclei isolation for Micro-dissected Mouse Brain Tissue . protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2zkpg5d/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: October 14, 2022
Last Modified: December 01, 2025
Protocol Integer ID: 71351
Keywords: seq nuclei isolation for micro, seq nuclei isolation, protocol for nuclei isolation, dissected mouse brain tissue, nuclei isolation, mouse brain tissue, storage of nuclei, brain regions from mice, dissected brain region, nuclei, seq library preparation, split
Abstract
Protocol for nuclei isolation prior to SPLiT-seq library preparation. Specifically optimized for the storage of nuclei from micro-dissected brain regions from mice or human (50-100mg).
Guidelines
Tissue collection for this protocol requires prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Materials
Dounce Homogenizers:
2mL x 60 mm KIMBLE Dounce tissue grinder set (Sigma-Aldrich, D8938)
Consumables:
DNA LoBind® Tubes, 2.0 mL, PCR clean, colorless (Eppendorf, 022431048)
DNA LoBind® Tubes, 1.5 mL, PCR clean, colorless (Eppendorf, 022341021)
SureStrain™ Premium Cell Strainers, 40 µM (MTC Bio, C4040)
Reagents:
1M CaCl2 in H2O (Sigma-Aldrich, 21115)
Mg(Ac)2 (Sigma-Aldrich, M5661)
0.5M EDTA, pH 8 (Invitrogen Ambion, AM9260G)
1M Tris-HCL, pH 8, Molecular Biology Grade, Ultrapure (ThermoFisher Scientific, J22638)
Triton X-100, laboratory grade (Sigma-Aldrich, X100)
Bovine Serum Albumin Fraction V, 7.5% solution (ThermoFisher Scientific, 15260037)
SUPERase•In™ RNase Inhibitor, 20 U/uL (Invitrogen, AM2696)
RNaseOUT™ Recombinant Ribonuclease Inhibitor, 5,000 units (Invitrogen, 10777019)
Troubleshooting
Setup
30m
Chill dounce homogenizers and tubes at 4°C in wet ice bucket
Per sample:
2mL x 60 mm KIMBLE Dounce tissue grinder set
(2) DNA LoBind® Tubes, 1.5 mL
Prepare stock lysis buffer (can be stored at 4°C for 1 week)
For 15 mL stock:
1.641 g Sucrose
75 µL 1M CaCl2
45 µL 1M Mg(Ac)2
3 µL 0.5M EDTA
150 µL Tris-HCl, pH 8.0
15 µL Triton X-100
Fill to 15 mL with UltraPure H2O (DNase-free, RNase-free)
Prepare wash & resuspension buffer
For 12 mL:
1.2 mL Bovine Serum Albumin Fraction V, 7.5% solution
120 µL SUPERase•In™ RNase Inhibitor, 20 U/uL
120 µL RNaseOUT™ Recombinant Ribonuclease Inhibitor, 5,000 units
Fill to 12 mL with 1X PBS
Dounce Homogenization
Add DTT and RNase inhibitors to lysis buffer
Aliquot 1 mL of stock lysis buffer per sample (~100 mg)
Per 1 mL (per sample)
Add 1:1000 1,4-Dithiothreitol (DTT)
Add 5 µL SUPERase•In™ RNase Inhibitor, 20 U/uL per mL
Add 5 µL RNaseOUT™ Recombinant Ribonuclease Inhibitor, 5,000 units
5m
Add 500 µL of lysis buffer (+ DTT & RNase inhibitors) to each sample (in a 1.5 mL eppendorf tube or cryovial)
5m
Incubate for 10 minutes on ice.
- Incubation time may need to be optimized depending on tissue
10m
With a wide-pore P1000 barrier pipet tip homogenize the tissue by pipetting up and down (3-5X) until the tissue readily passes through the pipet tip.
5m
Transfer tissue homogenate to the dounce homogenizer with a wide-pore P1000 barrier pipet tip.
5m
Wash the sample tube with of lysis buffer (+ DTT & RNase inhibitors) and transfer buffer to the dounce homogenizer.
- This step ensures optimal retention of semi-homogenized tissue for dounce homogenization
5m
Dounce 10-30 times with Pestle A
- The # of dounces should be optimized by tissue
10m
Dounce 5-10 times with Pestle B
- The # of dounces should be optimized by tissue
10m
Washing
Pass 500 µL at a time through a 40 µM strainer into a 1.5 mL LoBind Eppendorf tube (pre-chilled) using a wide-pore P1000 barrier pipet tip.
Note
When pipetting nuclei suspension through the strainer, make a seal between the pipet tip and the mesh, slowly pipet to pass nuclei through. (add picture)
Centrifuge at 600 x g for 10 min. @ 4°C.
- The spin speed can be adjusted between 300 - 800 x g, faster spins risk damage to the nuclear membrane.
Remove the supernatant and add 500 µL of wash & resuspension buffer. Let the pellet sit for 2 min. on ice prior to re-suspending.
Gently re-suspend the nuclei pellet with a wide-pore P1000 barrier pipet tip.
Centrifuge at 600 x g for 5 min. @ 4°C.
Repeat steps 14-16.
Note
The number of washes can be optimized by tissue type. Increased washes = less debris, however too many washes will negatively impact the final nuclei yield.
Start with 2-3 washes.
Remove the supernatant and add 500 µL of wash & resuspension buffer. Let the pellet sit for 2 min. on ice prior to re-suspending.
Gently re-suspend the nuclei pellet with a wide-pore P1000 barrier pipet tip.
Pass the resuspended nuclei through a 40 µm Flow cell strainer into a 1.5 mL LoBind Eppendorf tube (pre-chilled).
Centrifuge at 600 x g for 5 min. @ 4°C.
Remove the supernatant and add 750 µL of wash & resuspension buffer. Let the pellet sit for 2 min. on ice prior to re-suspending.
Note
At this point you can pause and check the quality of the nuclei under a microscope at 40-60X magnification.
Fixation and Permeabilization
Prepare solutions for fixation & permeabilization and place on ice:
Fixation Solution:
750 µL 16% Formaldehyde
2.25 mL 1X PBS
Permeabilization Solution:
2 mL 5% Triton X-100
2 µL SUPERase•In™ RNase Inhibitor, 20 U/uL
Neutralization Buffer:
500 µL 1M Tris-HCl, pH 8.0
200 µL 5% Triton X-100
4.8 mL 1X PBS
Filter nuclei through a 40 µM strainer into a 1.5 mL LoBind Eppendorf tube (pre-chilled) using a wide-pore P1000 barrier pipet tip to a 15 mL conical tube.
Note
This filter step is to ensure that only singlet nuclei are taken into the fixation. If there are a large presence of doublet nuclei in the sample this will lead to a high prevalence of doublets in the downstream dataset.
Add 250 µL of pre-chilled Fixation Solution and immediately mix gently by pipetting up and down 3X with a wide-pore P1000 barrier pipet tip.
Note
Caution should be taken when pipetting nuclei in the fixation solution, too forceful or frequent pipetting will risk shearing of nuclear membrane. Only pipet enough to suspend nuclei in solution (3X recommended)
Incubate for 10 minutes on ice.
Add 40 µL of Permeabilization Solution and mix gently by pipetting up and down 3X with a wide-pore P1000 barrier pipet tip.
Incubate for 3 minutes on ice.
Add 1 mL cold nuclei neutralization buffer and gently invert the tube 3X to mix
Centrifuge at 500 x g for 5 min. @ 4°C in the swinging bucket rotator.
Nuclei Storage
Prepare nuclei storage buffer and place on ice:
Nuclei Storage Buffer:
6 mL 1X PBS
15 µL SUPERase•In™ RNase Inhibitor, 20 U/uL
7.5 µL RNaseOUT™ Recombinant Ribonuclease Inhibitor, 5,000 units
Remove the supernatant and add 300 µL of Nuclei Storage Buffer. Let the pellet sit for 2 min. on ice prior to re-suspending.
Gently re-suspend the nuclei pellet with a wide-pore P1000 barrier pipet tip.
Filter nuclei through a 40 µM strainer into a 1.5 mL LoBind Eppendorf tube (pre-chilled) using a wide-pore P1000 barrier pipet tip.
Add 5 µL of DMSO and gently flick the tube 3X, wait 1 minute on ice. Repeat 3X to add a total of 15 µL DMSO.
Mix the final suspension 5X with a P200 barrier pipet tip and aliquot for future experiments.
Place aliquoted nuclei suspensions in a Mr. Frosty to cool at -1°C/minute in a -80°C freezer overnight.
Fixed nuclei suspensions can be stored at -80°C for up to 6 months.