Dec 12, 2023
Version 1
JAX - Nuclei Isolation for 10x Genomics A V.1
- William F Flynn1,
- Elise Courtois1,
- Greggory A Perry1,
- Sandra L Daigle1,
- Paul Gabriel1,
- Diane Luo1,
- Jessica Grassmann1
- 1The Jackson Laboratory
Protocol Citation: William F Flynn, Elise Courtois, Greggory A Perry, Sandra L Daigle, Paul Gabriel, Diane Luo, Jessica Grassmann 2023. JAX - Nuclei Isolation for 10x Genomics A. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1n9npgr2/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: March 14, 2022
Last Modified: December 12, 2023
Protocol Integer ID: 59445
Keywords: nuclei isolation, 10x genomics, multiome
Abstract
The purpose of this protocol is to produce single nuclei from frozen human tissues for downstream assaying with the 10x Genomics Multiome assay.
This protocol has been demonstrated using Human placenta tissue as well as human Glioblastoma.
This protocol is modified from Sigma Aldrich Nuclei Isolation Kit: EZ Prep protocol and 10x Chromium Demonstrated protocol for Single Cell Multiome ATAC + Gene Expression Sequencing (CG000365).
Guidelines
- Assess nuclear membrane integrity (refer to 10x Genomics CG000365), using Brightfield or Trypan blue
- Assess for debris, if large amount of debris present, filter with smaller pore size to decrease debris.
- Use either Trypan blue and Countess (for clean average size nuclei preps) or AO/PI and Nexcellom K2 counter (if nuclei prep has debris)
- If RNA content is of concern Imaging nuclei can be done with fluorescence, and Syto RNA stain
Materials
Nuclei Buffer (20x): 2000153/2000207 (10x Genomics)
Digitonin: BN2006 (Thermo Fisher)
Trizma Hydrochloride Solution, pH 7.4: T2194 (Sigma Aldrich)
Magnesium Chloride Solution: M1028 (Sigma Aldrich)
Sodium Chloride solution, 5M: 59222C (Sigma Aldrich)
1M Nonidet P40 substitute:74385(Sigma Aldrich)
Protector RNase inhibitor (DO NOT SUBSTITUTE): 3335399001 (Sigma Aldrich)
Nuclei Isolation Kit: Nuclei EZ Prep: NUC101-1KT (Sigma Aldrich)
DTT: 43816-10ML (Sigma Aldrich)
Tween-20: 1662404 (Bio-Rad)
PBS: 14190-144(Gibco)
BSA: 130-091-376 (Miltenyi)
Countess Slides: C10228 (Invitrogen)
Flowmi 40um filter (Bel-Art™ H136800040)
Bel-art disposable pestle: BAF199230000-100EA (Sigma Aldrich)
Pluristrainer mini filters: 43-10040-40 (Pluriselect)
Buffers
| A | B | C | D | |
| Stock | Final | 1mL | ||
| EZ lysis | - | - | 972ul | |
| Digitonin | 5% | 0.01% | 2ul | |
| DTT | 1000 mM | 1 mM | 1 μl | |
| RNase inhibitor 40 U/μl | 40 U/μl | 1 U/μl | 25ul |
Lysis Buffer, Prepare fresh, maintain at 4°C
| A | B | C | D | |
| Stock | Final | 4mL | ||
| Tris-HCl (pH 7.4) | 1 M | 10 mM | 40 μl | |
| NaCl | 5 M | 10 mM | 8 μl | |
| MgCl2 | 1 M | 3 mM | 12 μl | |
| Tween-20 | 10% | 1.00% | 400 μl | |
| BSA | 10% | 0% | 40 μl | |
| DTT | 1000 mM | 1 mM | 4 μl | |
| RNase inhibitor 40 U/μl | 40 U/μl | 1 U/μl | 100 μl | |
| Nuclease-free Water | - | - | 3.40 ml |
Wash Buffer, Prepare fresh, maintain at 4°C
| A | B | C | D | |
| Stock | Final | 1mL | ||
| Nuclei Buffer* (20X) | 20X | 1X | 50 μl | |
| DTT | 1000 mM | 1mM | 1 μl | |
| RNase inhibitor | 40 U/μl | 1 U/μl | 25 μl | |
| Nuclease-free Water | - | - | 924 μl |
Nuclei Buffer (1X), Prepare fresh, maintain at 4°C
Before start
Familiarize yourself with 10x Genomics protocol CG000365.
Sample Prep
Sample Prep
20m
20m
Slightly thaw sample out on ice and place onto pre chilled Petri dish on ice.
Cut tissue into small pieces and add to a microcentrifuge tube.
Cell lysis
Cell lysis
17m
17m
In micro centrifuge tube containing the tissue add 100 µL lysis buffer of lysis buffer.
Using a plastic pestle to grind tissue by pushing pestle down and twisting to break up the tissue.
Start timer once lysis buffer is added.
2m
Add 25 µL lysis buffer to wash off pestle and place tube On ice for a total of 00:05:00 .
5m
After 5 minutes of lysis On ice , 500 x g, 4°C, 00:05:00 .
5m
Remove supernatant and resuspend pellet using wide bore tips in 100 µL lysis buffer .
Incubate On ice for a total of 00:05:00 .
5m
Washing
Washing
15m
15m
Add 500 µL wash buffer and 500 x g, 4°C, 00:05:00 .
5m
go to step #9 Repeat for 3 total washes.
10m
After last wash, filter suspension through 40um filter.
Resuspend sample in 1x Nuclei buffer (10x provided) containing RNase inhibitor, and DTT.
Load
Load
5m
5m
Load for transposition and continue with the 10x multiome protocol.