Apr 28, 2026

10x Multiome (snRNA/ATACseq) of HuBMAP MOSDAP Tissues

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwwrbwvmk/v1
  • 1University of California San Diego
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: [email protected]
Scott Lindsay-Hewett
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvwwrbwvmk/v1
Protocol Citation: Scott Lindsay-Hewett, Tyler Ostrander, Abbas Hakim, Louise C. Laurent 2026. 10x Multiome (snRNA/ATACseq) of HuBMAP MOSDAP Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwwrbwvmk/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: April 28, 2026
Last Modified: April 28, 2026
Protocol  Integer ID: 315862
Keywords: Multiome, 10x Genomics, single nucleus, snATACseq, snRNAseq, 10x multiome, 10x multiome data from human biomolecular atlas program, hubmap mosdap tissues this protocol, hubmap mosdap tissue, 10x multiome data, human biomolecular atlas program, 10x multiome, quality templates for simultaneous atac, gene expression, multiple organ same donor authorization project, reverse transcription, following reverse transcription, simultaneous atac, atacseq, hubmap, bulk transposition, gem generation, nuclear suspension, human tissue, quality nuclear suspension
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 workflow used to generate 10x Multiome data from Human BioMolecular Atlas Program (HuBMAP) Multiple Organ Same Donor Authorization Project (MOSDAP) human tissues, beginning from high-quality nuclear suspensions prepared as described in the preceding protocol. It covers bulk transposition and GEM generation on the Chromium X. Following reverse transcription and quenching, the workflow concludes with GEM cleanup and pre-amplification, yielding high-quality templates for simultaneous ATAC and gene expression (GEX) library construction.
Guidelines
Use only 10x Genomics-validated emulsion-safe plastics.
Materials
Equipment

Vortex
Picofuge
Microfuge
Plate spinner
Thermal Cycler (BioRad cat # C1000 Touch with 96–Deep Well Reaction Module); others okay
Chromium X (or Chromium Controller)

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)
Reagent reservoirs, 10x approved, emulsion-safe (VWR cat # 41428-958)
DNA LoBind Tubes, 1.5 ml (Eppendorf cat # 022431021)
Low TE Buffer (Fisher cat # 12090-015)
Sigma Protector RNase Inhibitor (Sigma  cat # 3335399001)
USB Dithiothreitol (DTT), 0.1M Solution (Fisher cat # 707265ML)
Chromium Next GEM Chip J Single Cell Kit, 48 rxns (10x Genomics, PN-1000234)
10% Tween 20 (BioRad cat # 1662404)
Glycerin (glycerol), 50% (v/v) Aqueous Solution (Ricca Chemical Company cat # 3290-32)
TempAssure PCR 8-tube strip  (USA Scientific cat # 1402-4700)
Vortex Adapter (10x Genomics cat # 330002)
Chromium Next GEM Secondary Holder (10x Genomics cat # 3000332)
Nuclease-free Water (Fisher cat # AM9937)
200-proof ethanol
Qiagen Buffer EB (Qiagen cat # 19086)
SPRIselect Reagent Kit (Beckman Coulter cat # B23318)
Magnetic Separator/Magnetic Separator B (10x Genomics cat # 230003/2001212)
Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 16 rxns (10x Genomics cat # PN-1000283)
Nuclei Isolation & QC
Start with high-quality nuclei isolated according to the 10x Genomics Demonstrated Protocol (CG000375), and enriched using Miltenyi Biotec Anti-Nucleus MicroBeads, as detailed here:

Protocol
NAME

Nuclei Isolation from HuBMAP MOSDAP Tissues for 10x Multiome (snRNA/ATACseq)

CREATED BY
Scott Lindsay-Hewett

Bulk Transposition
Adjust the nuclei concentration to 3,220 nuclei/µL. Use 5 µL for the transposition reaction, as per the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide (CG000338).Download CG000338_ChromiumNextGEM_Multiome_ATAC_GEX_User_Guide_RevG.pdfCG000338_ChromiumNextGEM_Multiome_ATAC_GEX_User_Guide_RevG.pdf

Note
An initial input of 16,100 nuclei is targeted to achieve a recovery of approximately 10,000 nuclei post-processing.

After transposition, proceed immediately to GEM generation.
GEM Generation & Barcoding
Load a Chromium Next GEM Chip J (up to 8 samples per chip). Run the Chromium X (or Controller) to generate GEMs. Post-run, transfer GEMs to an 8-strip tube and proceed immediately to reverse transcription (RT).
Reverse Transcription & Quenching
Perform RT in a thermal cycler. Following RT, add Quenching Agent to stop the reaction. At this stage, samples may be stored at -80°C for up to 4 weeks.
GEM Cleanup & Pre-amplification PCR
To minimize batch effects, it is recommended to process all experimental samples simultaneously. Perform GEM cleanup and pre-amplification PCR (7 cycles).
  • This yields 160 µL of product containing barcoded transposed DNA and barcoded cDNA.
  • Aliquot for Library Construction: Use 40 µL for ATAC Library Construction and 35 µL for Gene Expression (GEX) Library Construction.
  • Store the remaining pre-amplification product at −20°C for future use.
Library Construction
Refer to the following protocols:

ATAC Library Construction:
Protocol
NAME

10x Multiome ATAC Library Construction and Sequencing

CREATED BY
Scott Lindsay-Hewett

GEX Library Construction

Protocol
NAME

10x Multiome Gene Expression Library Construction and Sequencing

CREATED BY
Scott Lindsay-Hewett