Nov 08, 2023
Protocol to isolate and fix nuclei from flash frozen female mouse gonads for IGVF
This protocol is a draft, published without a DOI.
- 1University of California, Irvine
Protocol Citation: Elisabeth Rebboah 2023. Protocol to isolate and fix nuclei from flash frozen female mouse gonads for IGVF. protocols.io https://protocols.io/view/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4p6yvre
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: November 07, 2023
Last Modified: November 08, 2023
Protocol Integer ID: 90590
Abstract
This protocol describes isolation of nuclei from left and right 10 week old mouse ovary and oviduct (pooled tissue ID: 26) from 8 founder strains (B6J, AJ, 129S1J, NZOJ, WSBJ, NODJ, PWKJ, and CASTJ), preparation of a single nucleus suspension, and fixation for single nucleus RNA-seq using Parse Biosciences. We process 1 rep from each strain per day; e.g. female rep 1 across all 8 strains. The main products we use are Parse Biosciences Nuclei Fixation Kit (v2) and Miltenyi Biotec’s gentleMACS Octo Dissociator with accessories. This protocol takes about 3.5 hours from start to finish.
The results are 2 aliquots of fixed single-nucleus suspensions for Parse per each of the 8 samples at >= 2,500 nuclei/ul.
The first part of the protocol describes tissue lysis and nuclei extraction using Miltenyi Biotec’s gentleMACS Octo Dissociator with accessories. The second part describes nuclei fixation using Parse Biosciences Evercode Nuclei Fixation Kit with v2 reagents. Due to low nuclei recovery, we modify the original Parse Biosciences Evercode fixation protocol (attached) by using half volumes of all fixation reagents. We do not fix extra nuclei for other assays such as SHARE-seq.
Attachments
Guidelines
- We recommend using a 5 ml pipette for aspirations and resuspensions > 1 ml.
Materials
| Name | Manufacturer | Cat. # | |
| Nuclei Fixation Kit v2 | Parse Biosciences | ECF2003 | |
| 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 | |
| gentleMACS C Tube | Miltenyi Biotec | 130-093-237 | |
| gentleMACS Octo Dissociator | Miltenyi Biotec | 130-095-937 | |
| MACS SmartStrainers (30 um) | Miltenyi Biotec | 130-098-458 | |
| NucBlue Fixed Cell ReadyProbes | Thermo Fisher | R37606 | |
| Hemacytometer | Fisher Scientific | 02-671-51B | |
| Mr. Frosty | Sigma-Aldrich | 635639 |
Reagents/equipment, manufacturer and catalog number
| Name | Reagent | Volume (for 8 samples) | Final Concentration | |
| Lysis buffer | Nuclei Extraction Buffer | 35 ml | NA | |
| 40 U/ul RNase inhibitor | 175 ul | 0.2 U/ul | ||
| NB-BSA + RNase inhibitor | Nuclei Buffer (Parse Biosciences) | 3.15 ml | NA | |
| 7.5% BSA | 350 ul | 0.75% | ||
| RNase inhibitor (Parse Biosciences) | 44.1 ul |
Buffers
Setup
Setup
Label tubes.
Pre-chill centrifuge to 4C.
Prepare ice buckets.
Prepare 35 ml lysis buffer on ice in a 50 mL conical tube. Distribute 2 mL into 8 gentleMACS C Tubes on ice. Add 175 ul RNase inhibitor to the lysis buffer aliquot the day of the experiment.
Prepare 3.5 ml NB + BSA. Add 44.1 ul RNase inhibitor included in Parse Biosciences fixation kit the day of the experiment.
Prepare 1.5 ml nuclei buffer + RNase inhibitor for final resuspension. Add 18.6 ul RNase inhibitor to 1.5 ml nuclei buffer.
Thaw components of 1 Parse Biosciences Nuclei Fixation v2 kit at room temperature, then place on ice.
Distribute 20 ul NucBlue Fixed Cell ReadyProbes into 16 PCR strip tubes for cell counting. Need 8 tubes for counting after nuclei extraction, and another 8 tubes for final fixed nuclei.
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. There should be 4 pieces: left and right ovary and left and right oviduct.
Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (~5 minutes).
Remove tubes, ensuring tissue did not get stuck on the sides, and spin down in a 4C centrifuge for ~10 seconds to bring liquid to the bottom, then place tubes back on ice.
Filter nuclei suspension through 30 um MACS SmartStrainer into a 5 ml tube. Fit a tube rack in ice for extra stability while filtering.
Wash 30 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 4 ml nuclei suspension at 4C, 350g for 5 minutes.
Discard supernatant and resuspend nuclei pellet in 750 ul NB-BSA + RNase inhibitor and filter through a 40 um strainer (provided in Parse Biosciences kit) into a new 5 ml tube. In our experience, total number of nuclei from female gonads is around 4 million.
Count nuclei. Use 1:6 dilution factor, 4 ul + 20 ul dye.
Parse Nuclei Fixation
Parse Nuclei Fixation
Add 250 uL Nuclei Fixation Solution and mix 3 times. Do not over-mix.
Incubate nuclei for 10 minutes on ice. Set 1 P200 pipette to 80 ul and keep the P1000 at 250 ul.
Add 80 uL Nuclei Permeabilization Solution and mix by pipetting 3 times with the P1000 still set to 250 uL. Do not over-mix.
Incubate 3 minutes with nuclei on ice.
Add 4 ml Nuclei Neutralization Solution and invert the tube once to mix.
Centrifuge at 4C, 750g for 10 minutes.
Aspirate and discard supernatant.
Resuspend the samples that started with < 2 million nuclei in 150 ul Nuclei Buffer with RNase inhibitor without BSA and transfer to a labeled 1.5 ml tube. Do NOT filter, will lose too much volume. For samples starting with > 2 million nuclei, resuspend in 300 ul Nuclei Buffer and filter.
Count nuclei. Use a 1:11 dilution factor, e.g. 2 ul + 20 ul dye.
Add Nuclei DMSO: 2.5 ul into 150 ul samples and gently flick tubes to mix. One minute later, add another 2.5 ul and flick to mix, then after another minute add a final 2.5 ul for a total volume of 7.5 ul. Mix by gently pipetting 5x with a P200 set to 75 ul.
Split nuclei suspension into 2 aliquots of equal volume in labeled 1.5 ml tubes. (E.g. if resuspend with 150 ul, split nuclei suspension into 75 ul per tube.)
Place tubes in a Mr. Frosty at -80C. The next day, move tubes to boxes in -80C racks.
Storage of leftover nuclei
Storage of leftover nuclei
Move remaining nuclei in RSB on ice to labeled 2 ml tubes.
Spin 750g, 4C, 5 minutes.
Remove all supernatant and flash-freeze nuclei as a dry pellet in liquid nitrogen. Store at -80C.