Oct 16, 2025
Human coronary nuclear isolation protocol
- Daniel Yuhang Li1,
- João Pinho Monteiro1
- 1Stanford University
Protocol Citation: Daniel Yuhang Li, João Pinho Monteiro 2025. Human coronary nuclear isolation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmbqd5g3p/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: October 14, 2025
Last Modified: October 16, 2025
Protocol Integer ID: 229812
Keywords: human coronary nuclear isolation protocol, human coronary nuclear isolation protocol this protocol, quality nuclei from human coronary tissue, clean nuclei suitable for fac, preservation of nuclear integrity, yielding clean nuclei, human coronary tissue, nuclear integrity, quality nuclei, 10x genomics capture, nucleus application
Abstract
This protocol outlines a streamlined method for isolating high-quality nuclei from human coronary tissue for downstream single-nucleus applications. The approach ensures efficient lysis, removal of debris, and preservation of nuclear integrity, yielding clean nuclei suitable for FACS and 10x Genomics capture.
Troubleshooting
Buffers
Nuclear buffer (Premade and stored at 4C)
- 500mL H20
- 1mL NaCl (5M) for final concentration 10mM
- 1.5mL MgCl (1M) for final concentration 3mM
- 5mL Tris-HCI (1M) for final concentration 10mM
10% BSA
- 10% BSA solution (Sigma-Aldrich, A1595-50ML)
Lysis buffer (1mL)
- 1uL NP-40 (1:1000) for final concentration 0.1%
- 10uL 10% BSA (1:100)
- 2uL 5% Digitonin (1:1000)
- 1uL Spermidine (500 mM stock) for final concentration 0.5mM
- 1uL Spermine (150 mM stock) for final concentration 0.15mM
- 25uL Protector RNase Inhibitor (40 U/uL stock) for final concentration 1 U/uL
Wash buffer (1mL)
- 975uL Nuclei buffer
- 10uL 10% BSA
- 25uL Protector RNase Inhibitor (40 U/uL stock) for final concentration 1 U/uL
1x nuclei buffer (1mL)
- 950uL H2O
- 10uL 10% BSA
- 50uL 20x nuclei buffer from 10x kit
Protocol
Cut a 30-40mg piece of tissue on a clean metal block cooled to freezing temp over dry ice. Keep sectioned and weighed tissue in 1.5ml Lo Bind tube on dry ice until ready to proceed.
NOTE: Large pieces of tissue >60mg will likely be underlysed and block the filters.
Place on ice, add 500uL of lysis buffer and finely mince tissue with small iris scissors (4 min).
Timing can be adjusted depending on tissue size, for 30-40mg this was sufficient.
Gently vortex and rest on ice for 5 min with intermittent vortexing.
Stop excess lysis by diluting in 500uL of wash buffer and proceed with filtering steps.
Filter through 70um strainer into new 1.5uL Lo Bind tube coated with BSA.
May need gentle centrifugation to move lysate through filter.
Repeat filtering through 10um strainer into new 1.5uL Lo Bind tube coated with BSA.
Discard strainer and add 500uL of wash buffer to further dilute detergent.
Centrifuge nuclei at 500xg for 10min at 4C
Remove supernatant and add 150uL of "10x nuclei buffer" without disturbing the pellet.
Centrifuge nuclei at 500xg for 5min at 4C and remove all supernatant (buffer exchange)
Resuspend in 7-30uL of 1x nuclei buffer.
FACS sorting prior to capture aim for at least 200k nuclei total and resuspend in ~500uL of chilled Wash Buffer (w/ BSA).
Access nuclei quality and count with Trypan blue.
Proceed to 10X capture.