Feb 01, 2025
Protocol to isolate and fix nuclei from flash frozen mouse brain for IGVF SHARE-seq
- 1University of California, Irvine
- IGVF
External link: https://static.miltenyibiotec.com/asset/150655405641/document_fks46sfs0p7c79hgi64bqfa33d?content-disposition=inline
Protocol Citation: Elisabeth Rebboah 2025. Protocol to isolate and fix nuclei from flash frozen mouse brain for IGVF SHARE-seq . protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8dq29g2w/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: January 31, 2025
Last Modified: February 01, 2025
Protocol Integer ID: 119404
Keywords: fixation, nuclei fixation, SHARE-seq, mouse brain, nuclei isolation, Mouse
Abstract
This protocol describes isolation of nuclei from brain tissues of 10-week-old B6J and CASTJ mice, preparation of a single nucleus suspension, selection for nuclei using Miltenyi Biotec’s Anti-Nucleus MicroBeads, and fixation for SHARE-seq using part of the Broad Institute protocol. We processed 8 samples at once (2 males and 2 females per strain) with two technicians in parallel. This protocol takes about 3 hours from start to finish. We have successfully tested this protocol with mouse brain cortex (either left or right side) and diencephalon (thalamus+hypothalamus)+pituitary.
Attachments
Guidelines
- Buffers can be prepared ahead of time and kept in 4°C. Add RNase inhibitors fresh the day of the experiment.
- We recommend using a 5 mL pipette for aspirations and resuspensions > 1 mL.
Materials
| Name | Manufacturer | Cat. # | |
| Nuclei Extraction Buffer | Miltenyi Biotec | 130-128-024 | |
| RNase Inhibitor, murine | New England Biolabs | M0314L | |
| PBS | HyClone | SH30256.02 | |
| 7.5% BSA | Life Technologies | 15260037 | |
| 1 M HEPES pH 7.3 | Sigma | H0887-100ml | |
| NaCl | Fisher | BP358-1 | |
| MgCl2 | Fisher | AA12315A7 | |
| Tween-20 | Fisher | BP337-500 | |
| 5% digitonin | Millipore Sigma | 300410 | |
| Enzymatics RI | Enzymatics | Y9240L | |
| SUPERase RI | Invitrogen | AM2696 | |
| Yeast tRNA | Invitrogen | AM7119 | |
| Glycine | Fisher | BP381-500 | |
| 1M Tris pH 8.0 | Thermo | AM9855G | |
| Formaldehyde (methanol-free) | EMS | 15710 | |
| gentleMACS C Tube | Miltenyi Biotec | 130-093-237 | |
| gentleMACS Octo Dissociator | Miltenyi Biotec | 130-095-937 | |
| MACS SmartStrainers (70 um) | Miltenyi Biotec | 130-110-916 | |
| MACS SmartStrainers (30 um) | Miltenyi Biotec | 130-098-458 | |
| LS Columns | Miltenyi Biotec | 130-042-401 | |
| QuadroMACS Separator | Miltenyi Biotec | 130-091-051 | |
| Anti-Nucleus MicroBeads | Miltenyi Biotec | 130-132-997 | |
| NucBlue Fixed Cell ReadyProbes | Thermo Fisher | R37606 | |
| Disposable hemocytometer | Millipore Sigma | MDH-4N1-50PK |
Buffers
Buffers
Prepare the following buffers:
| Reagent | Amount | Final concentration | |
| Nuclei Extraction Buffer | 35 mL | NA | |
| RNase inhibitor, murine | 175 uL | 0.2 U/uL |
Lysis buffer
| Reagent | Amount | Final concentration | |
| Nuclei Extraction Buffer | 35 mL | NA | |
| 7.5% BSA | 333 uL | 0.1% | |
| RNase inhibitor, murine | 125 uL | 0.2 U/uL |
RSB
| Reagent | Amount | Final concentration | |
| Nuclei Extraction Buffer | 6.9 mL | 14% | |
| PBS | 41.4 mL | NA | |
| 7.5% BSA | 257.6 uL | 0.04% |
Nuclei Suspension Buffer (NSB)
Make 2 tubes of 50 mL.
| Reagent | Amount | Final concentration | |
| Nuclei Suspension Buffer (NSB) | 30 mL | NA | |
| RNase inhibitor, murine | 150 uL | 0.2 U/uL |
NSB-RI
Make 2 tubes of 30 mL.
| Reagent | Amount | Final concentration | |
| H2O | 48.75 mL | NA | |
| 1 M HEPES pH 7.3 | 500 uL | 10 mM | |
| 5 M NaCl | 100 uL | 10 mM | |
| 1 M MgCl2 | 150 uL | 3 mM | |
| RNase inhibitor, murine | 500 uL | 0.1% |
HT-RSB
| Reagent | Amount | Final concentration | |
| HT-RSB | 25 mL | NA | |
| 7.5% BSA | 133.8 uL | 0.04% | |
| 5% digitonin | 50 uL | 0.01% | |
| Enzymatics RI | 62.5 uL | 0.1 U/uL | |
| SUPERase RI | 31.3 uL | 0.025 U/uL | |
| Yeast tRNA | 250 uL | 100 ug/mL |
HDT-2RI
Make 2 tubes of 25 mL.
| Reagent | Amount | Final concentration | |
| Glycine | 0.94 g | 2.5M | |
| H2O | 5 mL | NA |
2.5 M Glycine
Setup
Setup
Before starting, spray down the bench and pipettes with 70% ethanol and RNase-away.
Coat SHARE-seq nuclei prep tubes with BSA. Fill 1.5 mL tubes with 1.5 mL 1% BSA-DEPC and incubate for 30 minutes. After incubation, aspirate BSA solution and dry for 30 minutes. Store at 4°C. Alternatively, add 150 uL 7.5% BSA, vortex, remove supernatant, let tube sit on ice briefly, and aspirate BSA solution.
Pre-chill centrifuge to 4°C.
Prepare ice buckets.
Prepare 35 mL lysis buffer in a 50 mL conical tube on ice. Distribute 2 mL into 8 gentleMACS C Tubes on ice.
Prepare 25 mL RSB in a 50 mL conical tube on ice.
Prepare two 25 mL aliquots of HDT-2RI in 50 mL conical tubes. Keep one at room temperature and the other on ice.
Prepare two 40 mL aliquots of NSB in 50 mL tubes on ice.
Prepare a fresh 2.5M glycine solution (usable for up to one month). Weigh the glycine powder into a 5 mL tube, add water, and allow it to dissolve. If needed, incubate at 37°C in a thermomixer.
Distribute NucBlue Fixed Cell ReadyProbes into PCR strip tubes for cell counting.
Print labels and label 1.5 mL tubes for final fixed pellets.
Tissue lysis and nuclei extraction
Tissue lysis and nuclei extraction
Keep flash frozen tissue samples on dry ice until lysis.
Drop whole frozen tissue into a chilled gentleMACS C Tube with 2 mL lysis buffer. Close tubes firmly and invert immediately, ensuring tissue is not stuck to the bottom or side. Keep tubes on ice and proceed immediately to dissociation.
Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (~5 minutes). Add chilled adapters to make sure nuclei stay cold.
Remove tubes, ensuring tissue did not get stuck on the sides, and spin down in a 4°C centrifuge for ~10 seconds to bring liquid to the bottom, then place tubes back on ice.
Filter nuclei suspension through 70 um MACS SmartStrainer into a 5 mL tube. Fit a tube rack in ice for extra stability while filtering.
Wash 70 um MACS SmartStrainer with 2 mL additional lysis buffer. Add 2 mL to C tubes, cap, and swish to recover any nuclei stuck to the sides and cap of the C tubes, then wash the strainer.
Discard strainer and centrifuge the nuclei suspension at 4°C, 350g for 5 minutes.
Resuspend nuclei pellet in 3 mL RSB.
Filter nuclei suspension through 30 um MACS SmartStrainer into a labeled 5 mL tube.
Count nuclei. Use 1:11 dilution factor, 20 uL dye + 2 uL sample.
Anti-Nucleus MicroBeads selection
Anti-Nucleus MicroBeads selection
Set aside 4 million nuclei in RSB in a new 5 mL tube and spin down at 4°C, 300g for 5 minutes.
Pipette off supernatant completely without disturbing the nucleus pellet
Resuspend nucleus pellet in 1.8 mL NSB-RI.
(450 µL of nuclei separation buffer per 10⁶ total nuclei)
Add 200 uL of Anti-Nucleus MicroBeads
(50 µL of Anti-Nucleus MicroBeads per 10⁶ total nuclei)
Mix well and incubate for 15 minutes in the refrigerator (2−8°C).
During incubation, prepare LS columns (need 1 column per sample):
Place column in the magnetic field of a suitable MACS Separator.
- Label LS columns, 15 mL tubes for debris, and 5 mL tubes for nuclei.
- Place LS columns in the magnetic separator rack and 15 mL tubes in the clear plastic tube holder.
- Rinse the columns by passing 1 mL NSB (no RI) 3 times (3 mL total) with a 10 mL serological pipette. Wait the first mL pass through, no dripping, then add the next mL
Add 2 mL NSB-RI to the samples and proceed to magnetic separation with LS Columns.
Apply nucleus suspension onto the column, 1 mL at a time. Collect flowthrough containing debris in the 15 mL tubes. Add the nucleus suspension directly to the center of the column reservoir. Always wait until the column reservoir is empty before proceeding to the next step.
Wash column two times with 1 mL NSB-RI. Collect debris that passes through and combine with the flow-through from the previous step. Perform washing steps by adding buffer aliquots as soon as the column reservoir is empty. Use buffer to rinse the inner column walls.
Remove column from the separator and place it on a 5 mL collection tube. Typically just hold it over the 5 mL tube with one hand.
With the other hand, pipette 1 mL NSB-RI onto the column. Immediately flush out the magnetically labeled nuclei by firmly pushing the plunger into the column.
Optionally, count nuclei to measure recovery after bead cleanup. Start the 5 minute spin in the next section and count then; if necessary, finish during the 10 minute incubation at step 4 in the next section. Use 1:4 dilution factor (9 uL dye + 3 uL sample).
SHARE-seq nuclei fixation
SHARE-seq nuclei fixation
Spin down nuclei in the 5 mL collection tubes at 4°C, 750g for 5 minutes.
Remove supernatant and resuspend nuclei pellet in 4 mL room temperature HDT-2RI. Transfer tube to a room temperature rack.
At RT, add 53.4 uL of methanol-free formaldehyde in the fume hood (16% stock solution). Final concentration for nuclei: 0.2%. Close tube and nutate or rotate nuclei at RT for 5 minutes.
To quench fixation, per reaction, add 224.4 uL fresh 2.5M Glycine, 200 uL of 1M Tris pH 8.0, 53.2 uL of 7.5% BSA, and mix using a pipette. Incubate on ice for 10 minutes.
Spin 750g, 4°C, 5 minutes. Gently remove supernatant.
Add 500 uL of HDT-2RI and gently resuspend pellet. Transfer to labeled 1.5 mL tube. Store on ice.
a. Count nuclei at 1:10 dilution factor (18 uL dye + 2 uL sample)
b. For multiple aliquots, split 500 uL into 2 tubes of 250 uL.
Spin 750xg, 4°C, 10 minutes. Gently remove supernatant. Remove all fluid and freeze at -80C as a dry pellet.