Nov 08, 2023
Version 2
Protocol to isolate and fix nuclei from flash frozen mouse hypothalamus and pituitary gland for IGVF V.2
This protocol is a draft, published without a DOI.
- 1University of California, Irvine
- IGVF
Protocol Citation: Elisabeth Rebboah 2023. Protocol to isolate and fix nuclei from flash frozen mouse hypothalamus and pituitary gland for IGVF. protocols.io https://dx.doi.org/Version created by Elisabeth Rebboah
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: 90558
Keywords: Fixation, Nuclei fixation, Split-seq, SHARE-seq, Evercode, snRNA-seq, Parse Biosciences, Hypothalamus, Pituitary, Hypothalamus/pituitary, Nuclei isolation, Mouse, Mouse brain, Mortazavi, IGVF, UCI, nuclei from flash frozen mouse hypothalamus, using parse biosciences evercode nuclei fixation kit, nuclei from flash frozen mouse, nucleus suspensions for parse, fixation for single nucleus rna, isolation of nuclei, single nucleus rna, single nucleus suspension, parse biosciences protocol, remaining nuclei, flash frozen mouse hypothalamus, nuclei fixation, nucleus suspension, fixed nuclei pellet, nuclei pellet, pituitary gland for igvf, nuclei, week old mouse hypothalamus, old mouse hypothalamus, using parse bioscience, nucleus, rep from each strain, parse bioscience, strains for share, seq fixation protocol, pituitary gland, flash frozen mouse, seq protocol, rna
Abstract
This protocol describes isolation of nuclei from 10 week old mouse hypothalamus and pituitary gland (tissue ID: 01) from 8 founder strains (B6J, AJ, 129S1J, NZOJ, WSBJ, NODJ, PWKJ, and CASTJ), preparation of a single nucleus suspension, and fixation for 1. single nucleus RNA-seq using the Parse Biosciences protocol (Split-seq) and 2. single nucleus RNA-seq + ATAC-seq using the SHARE-seq protocol. We process 1 rep from each strain per day; e.g. female rep 1 across all 8 strains. For 8 samples, 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, and 1 fixed nuclei pellet pooled across all 8 strains for SHARE-seq, all stored at -80C.
The first part of the protocol describes tissue lysis and nuclei extraction using Miltenyi Biotec’s gentleMACS Octo Dissociator with accessories. When nuclei are extracted and counted, we determine whether we have enough to fix for Split-seq and SHARE-seq and set aside 2-4 million and 1 million, respectively. Ideally, the second and third parts of this protocol are performed in parallel by at least two technicians to save time. The second part describes nuclei fixation using Parse Biosciences Evercode Nuclei Fixation Kit with v2 reagents (see attachment for original version). The third part describes nuclei fixation using a modified version of the SHARE-seq fixation protocol (see attachment for original version). Any remaining nuclei are flash-frozen as a dry pellet and stored at -80C.
Guidelines
- We recommend using a 5 ml pipette for aspirations and resuspensions > 1 ml.
- When possible, after nuclei isolation and during the first round of counting, 2 technicians should continue on with Parse fixation after establishing the volume needed for roughly 4 million nuclei per sample (4 samples processed per technician). The remaining technician should determine the exact volume needed for 1 million cells and proceed with SHARE-seq fixation. Parallel fixation saves about an hour of time.
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 | |
| 1 M HEPES pH 7.3 | Sigma | H0887-100ml | |
| NaCl | Fisher | BP358-1 | |
| MgCl2 | Fisher | AA12315A7 | |
| Tween-20 | Fisher | BP337-500 | |
| 5% digitonin | Promega | G944A | |
| 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 | |
| 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 | |
| 1% BSA-DEPC | BSA | 1 g | 1% | |
| DEPC water | 100 ml | NA | ||
| 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) | 7 ml | NA | |
| 7.5% BSA | 700 ul | 0.75% | ||
| RNase inhibitor (Parse Biosciences) | 35 ul | |||
| RSB | PBS | 24.6 ml | NA | |
| 7.5% BSA | 333 ul | 0.1% | ||
| RNase inhibitor | 125 ul | 0.2 U/ul | ||
| SHARE-RSB | 1 M HEPES pH 7.3 | 150 ul | 10 mM | |
| 5 M NaCl | 30 ul | 10 mM | ||
| 1 M MgCl2 | 45 ul | 3 mM | ||
| 10% Tween-20 | 150 ul | 0.1% | ||
| H2O | 14.625 ml | NA | ||
| 7.5% BSA | 80.26 ul | 0.04% | ||
| 5% digitonin | 30 ul | 0.01% | ||
| Enzymatics RI | 37.5 ul | 0.1 U/ul | ||
| SUPERase RI | 18.75 ul | 0.025 U/ul | ||
| Yeast tRNA | 150 ul | 100 ug/ml |
Buffers
Setup
Coat SHARE-seq nuclei prep tubes with BSA. Fill 8 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 4C.
Label tubes.
Pre-chill centrifuge to 4C.
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. Add 175 ul RNase inhibitor to the lysis buffer aliquot the day of the experiment.
Prepare RSB in a 50 ml conical tube on ice. Add RNase inhibitor 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 2.5 ml nuclei buffer + RNase inhibitor for final resuspension. Add 31.5 ul RNase inhibitor to 2.5 ml nuclei buffer.
Prepare 15 ml SHARE-RSB in a 50 ml conical tube at room temperature. To SHARE-RSB, add 30 ul digitonin, 37.5 ul Enzymatics RI, 18.75 ul SUPERase RI, and 150 ul yeast tRNA fresh the day of the experiment.
Thaw components of 2 Parse Biosciences Nuclei Fixation v2 kits 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
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).
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 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 4 ml nuclei suspension at 4C, 350g for 5 minutes.
Discard supernatant and resuspend nuclei pellet in 3 ml RSB.
Filter nuclei suspension through 30 um MACS SmartStrainer into a 5 ml tube.
Count nuclei. Use 1:3 dilution factor, 10 ul + 20 ul dye.
Parse nuclei fixation
Set aside 2-4 million nuclei in RSB in a new 1.5 ml tube and spin down at 4C, 350g for 5 minutes.
Remove supernatant and and resuspend nuclei 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.
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 in 200 ul Nuclei Buffer with RNase inhibitor without BSA and move through a 40 um filter into a labeled 1.5 ml tube.
Count nuclei. Use 1:11 dilution factor, e.g. 2 ul + 20 ul dye.
Add Nuclei DMSO: 3.3 uL and gently flick tubes to mix. One minute later, add another 3.3 uL and flick to mix, then after another minute add a final 3.3 uL for a total volume of 9.9 uL. Mix by gently pipetting 5x with a P200 set to 100 ul.
Split nuclei suspension into 2 labeled tubes, 100 ul per tube.
Place tubes in a Mr. Frosty at -80C. The next day, move tubes to boxes in -80C racks.
SHARE-seq nuclei fixation
Set aside 1 million nuclei for each of the 8 samples in RSB and spin down at 4C, 750g for 5 minutes.
Remove supernatant and resuspend nuclei pellet in 1 ml room temperature SHARE-RSB. Transfer tube to a room temperature rack.
At RT, add 13.34 ul of methanol-free formaldehyde (16% stock solution). Final concentration for nuclei: 0.2%. Close tube and nutate cells at RT for 5 minutes.
To quench fixation, per reaction, add 56.1 ul fresh 2.5M Glycine (0.94g per 5 ml stock), 50 ul of 1M Tris pH 8.0, 13.3ul of 7.5% BSA, and mix using a pipette. Incubate on ice for 10 minutes.
Spin 750g, 4C, 5 minutes. Gently remove supernatant.
Add 200 ul of SHARE-RSB and gently resuspend pellet. Store on ice until all samples are completed.
Pool 200 ul of resuspended nuclei from all 8 founders into 1 labeled 2 ml tube.
Spin 1,000g, 4C, 10 minutes. Gently remove supernatant. Remove all fluid and freeze at -80C as a dry pellet.
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.