Apr 28, 2026
Version 2
Nuclei Isolation from HuBMAP MOSDAP Tissues for 10x Multiome (snRNA/ATACseq) V.2
- Scott Lindsay-Hewett1,
- Tyler Ostrander1,
- Abbas Hakim1,
- Louise C. Laurent1
- 1University of California San Diego
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: [email protected]
Protocol Citation: Scott Lindsay-Hewett, Tyler Ostrander, Abbas Hakim, Louise C. Laurent 2026. Nuclei Isolation from HuBMAP MOSDAP Tissues for 10x Multiome (snRNA/ATACseq). protocols.io https://dx.doi.org/10.17504/protocols.io.261geymrjv47/v2Version created by Scott Lindsay-Hewett
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: April 28, 2026
Last Modified: April 28, 2026
Protocol Integer ID: 315925
Keywords: Multiome, 10x Genomics, single nucleus, snATACseq, snRNAseq, nuclei isolation, nuclei isolation from hubmap mosdap tissue, human tissues for the 10x multiome assay, hubmap mosdap tissue, 10x multiome assay, preparation of nuclear suspension, nuclear suspension, human biomolecular atlas program, nuclei isolation, 10x multiome, human tissue, multiple organ same donor authorization project, frozen tissue, hubmap, snrna, mosdap, atacseq
Funders Acknowledgements:
National Institutes Of Health - Female Reproductive Tissue Mapping Center
Grant ID: U54 HD104393
National Institutes Of Health - Pregnant Female Reproductive Tissue Mapping Center
Grant ID: U54 HD110347
Abstract
This protocol describes the preparation of nuclear suspensions from Human BioMolecular Atlas Program (HuBMAP) Multiple Organ Same Donor Authorization Project (MOSDAP) human tissues for the 10x Multiome assay. It is compatible with both snap-frozen tissue and cryosections of carboxymethyl cellulose (CMC)-embedded tissue.
Guidelines
- Prior to assay execution, store snap-frozen tissue in LN2 vapor and CMC-embedded tissue at -80 ºC for long-term preservation and optimal data quality.
- Incubate digitonin at 65 ºC to fully dissolve any precipitate before use.
- Keep all nuclei isolation buffers and samples strictly on ice.
- Use only 10x Genomics-validated emulsion-safe plastics.
Materials
Equipment
Vortex
Picofuge
Refrigerated microfuge
Microscope with 20x and 40x objectives (to assess nuclei quality)
BioRad TC10 cell counter
Materials/reagents
Rainin pipettes (single and multichannel) and filter tips (10x approved, emulsion-safe)
Corning 15 ml and 50 ml tubes, 10x approved, emulsion-safe (Corning cat # CLS430791 and CLS430829)
DNA LoBind Tubes, 1.5 ml (Eppendorf cat # 022431021)
DNA LoBind Tubes, 2.0 ml (Eppendorf cat # 022431048)
Sigma Protector RNase Inhibitor (Sigma cat # 3335399001)
Bovine Serum Albumin solution (Sigma cat # A1595-50ML)
USB Dithiothreitol (DTT), 0.1M Solution (Fisher cat # 707265ML)
Trypan Blue Label (0.4%) (Fisher cat # T10282)
Aluminum foil
Hammer
Dry ice
Spatulas
Digitonin (Thermo cat # BN2006)
Trizma Hydrochloride Solution, pH 7.4 (Sigma cat # T2194)
Sodium Chloride Solution, 5 M (Sigma cat # 59222C)
Magnesium Chloride Solution, 1M (Sigma cat # M1028)
IGEPAL CA-630 (Sigma cat # i8896)
Cordless motor (Fisher cat # K7495400000)
Bel-Art Disposable Pestles (Sigma cat # BAF199230000-100EA)
Pluristrainer Mini 20um 100/pk (Fisher cat # NC1423042)
Pluristrainer Mini 40 Um 100pk (Fisher cat # NC1469671)
Pluristrainer Mini 70um 100/pk (Fisher cat # NC1587227)
Thermo Scientific ART Wide Bore Filtered Pipette Tips (Fisher cat # 212362C and 212361A)
Cell Counting Slides for TC10 (BioRad cat # 1450015)
10% Tween 20 (BioRad cat # 1662404)
Nuclease-free Water (Fisher cat # AM9937)
Nuclei Isolation
Isolate nuclei either from snap-frozen tissue chunks, or from cryosections of tissue embedded in carboxymethyl cellulose (CMC), according to the 10x Genomics Demonstrated Protocol (CG000375) - "Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing".
CG000375_DemonstratedProtocol_NucleiIsolationComplexSample_ATAC_GEX_Sequencing_Rev_D.pdf
CG000375_DemonstratedProtocol_NucleiIsolationComplexSample_ATAC_GEX_Sequencing_Rev_D.pdf Note
- To maximize Chromium Chip occupancy (8 channels), utilize a two-person team to isolate nuclei from up to 8 samples:
- Staggered Processing: Each operator manages 4 samples, staggering start times by 5 minutes.
- Standardized Holding Point: If staggering, hold nuclei on ice after the second wash (post-lysis) until all samples are ready for simultaneous downstream processing.
The following optimizations are applied:
Cryo-pulverization
- Tissue Protection: Prepare double-wrapped aluminum foil pockets pre-chilled on a dry ice block.
- Pulverization: Place tissue into the pre-chilled pocket and seal securely to prevent sample loss. Using a hammer, pulverize the tissue on dry ice until a fine powder consistency is achieved.
- Transfer: Using a pre-cooled spatula, transfer the tissue powder into 300 µL of chilled NP40 lysis buffer.
Lysis and Homogenization
- Homogenization: Immediately homogenize the powder on ice using a disposable plastic pestle attached to a cordless motor. Transfer the homogenate to 1 mL of NP40 lysis buffer using a wide-bore pipette tip.
- Incubation: Incubate on ice for 5 minutes, pipetting the suspension several times to facilitate lysis.
Note
Incubation in lysis buffer is limited to 1 minute for lymph node and thymus tissue because of their immune-rich composition and sensitivity to over-lysis.
- Filtration: Pass the lysate through a 70 µm cell strainer and proceed with standard wash steps. Following the second wash, sequentially filter the nuclei through 40 µm and 20 µm strainers to ensure a high-quality single-nuclei suspension.
- Flow Cytometry: FACS/Flow sorting is bypassed in this optimized workflow; proceed instead to nuclei enrichment with Miltenyi Biotec Anti-Nucleus MicroBeads.
Nuclei Enrichment with Miltenyi Biotec Anti-Nucleus MicroBeads
This additional step is included to remove debris (which may vary depending on tissue type) and to enrich for high-quality nuclei for downstream steps.
Following filtration through a 20 µm strainer (as described in the previous section), centrifuge 500 rcf, 4°C, 00:05:00 and proceed according to the Miltenyi Biotec product manual.
Miltenyi_Anti-Nucleus_MicroBeads.pdf202.2KB In brief, resuspend the nuclei pellet in 450 µL Nuclei Separation Buffer (as specified in the product manual), add 50 µL Anti-Nucleus MicroBeads, mix thoroughly, and incubate for 15 minutes at 4 °C.
Note
To minimize additional handling steps, nuclei are not counted prior to resuspension. Volumes of Nuclei Separation Buffer and Anti-Nucleus MicroBeads are calculated assuming a maximum input of 1 × 10^6 nuclei for enrichment. This exceeds the number of nuclei required for the 10x Multiome assay, ensuring sufficient material is recovered even if additional nuclei are present but not captured.
Perform the subsequent magnetic labeling and magnetic separation steps as outlined in the product manual, eluting the enriched nuclei in 1 ml Nuclei Separation Buffer at the final step.
Centrifuge the enriched nuclei 500 rcf, 4°C, 00:05:00 , then proceed directly to nuclei permeabilization.
Nuclei Permeabilization and Resuspension, QC
Permeabilization
Permeabilize enriched nuclei according to steps outlines in the 10x Genomics Demonstrated Protocol (CG000375) described above.
Resuspension and Quantification
- Final Resuspension: Following permeabilization, resuspend the nuclei in 25–200 µL of Diluted Nuclei Buffer (adjust volume based on pellet size).
- QC & Counting: Quantify using a BioRad TC10 cell counter (or equivalent) with Trypan Blue staining.
Expected result
Nuclei should appear round and intact, with smooth membranes, minimal blebbing, and no clumping. The example shown here is from pancreas tissue.
Following QC and counting, proceed immediately to the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression assay, as detailed here: