Customer Developed Protocol‘Frankenstein’ protocol for nuclei isolation fromfresh and frozen tissue
Contributed by:

Luciano Martelotto, Ph.D., Melbourne, Centre for Cancer Research, VictorianComprehensive Cancer Centre

CUSTOMER DEVELOPED PROTOCOL‘Frankenstein’ protocol for nuclei isolation from fresh and frozen tissue

Contributor Research Profile
Dr. Luciano Martelotto leads the single-cell innovation team at the University of Melbourne, Centre for Cancer
Research, Victorian Comprehensive Cancer Centre. His team, in collaboration with Dr. Richard Tothill (Rare Diseases
Oncogenomic Lab) and Prof. Sean Grimmond (Centre Director), implements new single-cell technologies anddevelops new techniques and protocols.
Learn more about their research: https://mdhs.unimelb.edu.au/our-organisation/institutes-centresdepartments/umccr/research/our-research
The University of Melbourne, Centre for Cancer Research is part of an alliance whose members are The Universityof Melbourne, Peter MacCallum Cancer Centre, The Royal Melbourne Hospital, The Walter and Eliza Hall Institute ofMedical Research, The Royal Women’s Hospital, The Royal Children’s Hospital, Western Health, St Vincent’sHospital Melbourne, Austin Health and the Murdoch Children’s Research Institute.
Read more here: https://www.viccompcancerctr.org/References
This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq
experiments using droplet-based methods, hence the name Frankenstein. Developed to prepare nuclei isolates
from small sample sizes (as little as grain of rice), this protocol uses FACS to identify cell subpopulations basedon ploidy (e.g. tumor versus stroma), to ensure that nuclei suspensions are not clumped, and to remove any
debris, especially ambient RNA, to help reduce background. The reference protocols can be found in thefollowing papers: Hu, et al., Habib, et al. (2016), Habib, et al. (2017), Lake, et al., and Lacar, et al.
This protocol is routinely used in the single-cell innovation lab for single nuclei experiments using 10x Genomics
technologies.
The protocol has been demonstrated to work successfully with fresh, snap/flash frozen, cryopreserved cells, andcell lines, as well as various solid cancers: pancreas, pheochromocytomas, paragangliomas, breast cancer,
lymphoma, xenografts and tumors.
NOTE: The protocol did not work with cardiomyocytes due to the high level of myosin heavy chain (MHC).

10x Genomics Products
Chromium Single Cell Immune Profiling Solution - https://www.10xgenomics.com/solutions/vdj/
Chromium Single Cell Gene Expression Solution - https://www.10xgenomics.com/single-cell/

Customer Developed Protocol

COMMUNITY.10XGENOMICS.COM

CUSTOMER DEVELOPED PROTOCOL‘Frankenstein’ protocol for nuclei isolation from fresh and frozen tissue

Notices
THIS PROTOCOL IS NOT SUPPORTED BY 10X GENOMICSThis Customer Developed Protocol is provided for general information only and is not directly supported,
endorsed, or certified by 10x Genomics. 10x Genomics gives no warranties and makes no claims about theprovided protocol.
For questions or to share protocol adjustments, please visit the 10x Community at community.10xgenomics.com

Customer Developed Protocol

COMMUNITY.10XGENOMICS.COM

CUSTOMER DEVELOPED PROTOCOL‘Frankenstein’ protocol for nuclei isolation from fresh and frozen tissue

Protocol Overview
NOTE: This protocol requires access to a cell sorter and familiarity with sorting cells/nuclei into 96-well plates.
1.
2.
3.
4.

Use a plastic pestle to mechanically homogenize tissue and release nuclei
Separate the nuclei from debris using a cell sorter
Collect a specific number of nuclei in a 96-well plate containing 10x RT Buffer*
Immediately load the sample into a Single Cell Chip for processing according the Single Cell 3’ ReagentsUser Guide or Single Cell V(D)J 5’ Reagents User Guide.

*Assume that nuclei recovery is 57%, and use this to determine the number of nuclei to collect for each ofyour samples. (This value is derived from the Cell Suspension Volume Calculator Table in the Single Cell 3’Reagents User Guide or Single Cell V(D)J 5’ Reagents User Guide)

Customer Developed Protocol

COMMUNITY.10XGENOMICS.COM

CUSTOMER DEVELOPED PROTOCOL‘Frankenstein’ protocol for nuclei isolation from fresh and frozen tissue

Required Buffers and Reagents
1. Nuclei EZ Lysis Buffer (Millipore Sigma) (chilled, 4°C)
2. Nuclei wash and resuspension buffer (prepare chilled, 4°C)
1x PBS
1.0% BSA
0.2 U/μl RNase Inhibitor

3. Nuclei wash and resuspension buffer with DAPI (prepare chilled, 4°C)
1x PBS
1.0% BSA
0.2 U/μl RNase Inhibitor
10 ug/mL DAPI

4. 10x RT Buffer1 for Single Cell Gene Expression 3’ reagents (DO NOT add RT enzyme)
RT Reagent Mix: 50 uLRT primer: 3.8 uL
Additive A: 2.4 uL
H2O: (33.8 – X – Y) uL

5. 10x RT Buffer1 for Single Cell Immune Profiling 5’ reagents (DO NOT add RT enzyme)
RT Reagent Mix: 50 uL
RT primer: 5.9 uL
Additive A: 2.4 uL
H2O: (31.7 – X – Y) uL
1

RT Buffer Notes•

X (‘sorting volume’): In the cytometric analysis setup described in this protocol, each droplet is 1 nL.
Example: 10,000 nuclei = 10,000 nL = 10 uL ‘sorting volume’.

•Y (‘additional volume’): This accounts for any additional volume deposited by the flow cytometer nozzle.
In the cytometric analysis setup described in this protocol (i.e. 75 um nozzle) there is no additional
volume deposited by the nozzle, so Y = 0 If in doubt, or to be on the safe side, just make Y= 5-10 uL.

•The 1 nuclei/nL assumption was corroborated empirically by sorting 10,000 nuclei in ten wells
containing 70 uL PBS and then measuring the final volume post sorting. It is highly recommended todetermine X empirically as value may vary depending on different sorters/nozzle combinations. It isrecommended to determine it at least once.

•Always measure the volume after sorting and top up to 90 uL with PBS or H2O if required.

•After adding the RT Enzyme Mix the final volume will be 100 uL.

•It is crucial to work as fast as possible. Do not leave nuclei sitting on ice for too long (e.g. 30’ is too long).

•Reduce as much as possible the time from sorting-to-controller run, ideally keep it under 40 minutes.
The longer the time the higher the background will be.

Customer Developed Protocol

COMMUNITY.10XGENOMICS.COM

CUSTOMER DEVELOPED PROTOCOL‘Frankenstein’ protocol for nuclei isolation from fresh and frozen tissueProtocol
NOTE: All samples and reagents are kept on ice or at 4 °C (wet ice).
1. Mince/chop tissue with a razor blade to small pieces. The tissue may be as small as a grain of rice.
2. Add 500 uL chilled Nuclei EZ Lysis Buffer to the tissue in 1.5 mL tube. Homogenize the sample using adouncer (stroking ~10-20 times). For mincing the tissue, you may take the tube out of ice, however, bequick and return to ice.
3. Transfer the homogenate (~500 uL) into a 2 mL tube and add 1 mL of chilled Nuclei EZ Lysis Buffer, mix
gently and incubate on ice for 5 min. Gently mix with a wide bore tip and repeat 1-2 times during theincubation.
4. Filter homogenate using a 70 μm-strainer mesh. Collect flow through in a Polystyrene round-bottom
FACS tube and transfer volume back into a new 2 mL tube.
5. Centrifuge the nuclei at 500g for 5 minutes at 4°C and remove supernatant leaving behind ~50 uL.
Gently resuspend nuclei in another 1.5 mL of EZ Lysis buffer, incubate for 5 minutes on ice.
6. Centrifuge the nuclei at 500g for 5 min at 4°C, remove supernatant as much as possible without
disturbing pellet (if pellet looks loose leave ~50 uL behind) and add 500 uL Nuclei Wash andResuspension Buffer and incubate 5 minutes without resuspending to allow buffer interchange. Afterincubation, add 1 mL of Nuclei Wash and Resuspension Buffer and resuspend the nuclei.
7. Centrifuge the nuclei at 500g for 5 min at 4°C, remove supernatant leaving behind ~50 uL and gently
resuspend nuclei in 1.4 mL Nuclei Wash and Resuspension Buffer and transfer to a 1.5 mL tube (easier tosee small pellets).
8. Repeat 7 and resuspend in 500 uL Nuclei Wash and Resuspension Buffer supplemented with DAPI.
Collect all nuclei by washing off nuclei from the wall of centrifuge tube.
IMPORTANT: Protect from light from here forward.
9. Filter nuclei (at least once) with a 35-µm cell strainer. Visually inspect nuclei integrity under amicroscope and count the number of nuclei with a cell counter or hematocytometer if required.
10. Perform cytometric analysis. Identify single nuclei and sub-populations based on DNA content, gate andsort directly into 10x RT Buffer prepared without the RT Enzyme Mix.
11. Proceed immediately with the 10x Genomics Single Cell Protocol and minimize the time between nuclei
preparation/sorting and chip loading. Add 10 uL RT Enzyme Mix to the sorted nuclei in RT buffer, mix
well but gently and load chip as per the Single Cell 3’ Reagents User Guide or Single Cell V(D)J 5’Reagents User Guide.

THIS PROTOCOL IS NOT SUPPORTED BY 10X GENOMICSThis 10x Community Customer Developed Protocol is provided for general information only andis not directly supported, endorsed, or certified by 10x Genomics. 10x Genomics gives no warranties and makes
no claims about the provided protocol.
For questions or to share protocol adjustments, please visit the 10x Community at community.10xgenomics.com

Customer Developed Protocol

COMMUNITY.10XGENOMICS.COM