Feb 25, 2026

Public workspace10x Multiome (snRNA/ATACseq) of Human Placenta

DOI
https://dx.doi.org/10.17504/protocols.io.q26g779wkgwz/v1
  • Scott Lindsay-Hewett1,
  • Tyler Ostrander1,
  • Abbas Hakim1,
  • Morgan Meads1,
  • Valentina Stanley1,
  • Mana Parast1,
  • Louise C. Laurent1
  • 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.q26g779wkgwz/v1
Protocol Citation: Scott Lindsay-Hewett, Tyler Ostrander, Abbas Hakim, Morgan Meads, Valentina Stanley, Mana Parast, Louise C. Laurent 2026. 10x Multiome (snRNA/ATACseq) of Human Placenta . protocols.io https://dx.doi.org/10.17504/protocols.io.q26g779wkgwz/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: February 25, 2026
Last Modified: February 26, 2026
Protocol Integer ID: 243979
Keywords: Multiome, 10x Genomics, single nucleus, snATACseq, snRNAseq, placenta, nuclei isolation, 10x multiome workflow for human placenta, optimized 10x multiome workflow, 10x multiome, human placenta, quality templates for simultaneous atac, atacseq, simultaneous atac, reverse transcription, following reverse transcription, nuclei isolation, spanning nuclei isolation, gem generation, snrna, bulk transposition
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 outlines an optimized 10x Multiome workflow for human placenta, spanning Nuclei Isolation, Bulk Transposition, and GEM Generation on the Chromium X. Following Reverse Transcription and Quenching, the process concludes with GEM Cleanup and Pre-amplification, yielding high-quality templates for simultaneous ATAC and GEX library construction.
Guidelines
  • Prior to assay execution, store tissue in LN2 vapor for long-term preservation and optimal data quality.
  • Incubate digitonin at 65ºC 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
Plate spinner
Thermal Cycler (BioRad cat # C1000 Touch with 96–Deep Well Reaction Module); others okay
Chromium X (or Chromium Controller)
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)
Reagent reservoirs, 10x approved, emulsion-safe (VWR cat # 41428-958)
DNA LoBind Tubes, 1.5 ml (Eppendorf cat # 022431021)
DNA LoBind Tubes, 2.0 ml (Eppendorf cat # 022431048)
Low TE Buffer (Fisher cat # 12090-015)
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)
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)
Troubleshooting
Nuclei Isolation & QC
Isolate nuclei from snap frozen placenta tissue according to the 10x Genomics Demonstrated Protocol (CG000375) - "Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing".Download CG000375_DemonstratedProtocol_NucleiIsolationComplexSample_ATAC_GEX_Sequencing_Rev_D.pdfCG000375_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 placenta-specific optimizations are used:

Cryo-pulverization

  • Tissue Protection: Prepare double-wrapped aluminum foil pockets pre-chilled on a dry ice block.
  • Pulverization: Place snap-frozen placenta 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.
  • 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 directly to nuclei permeabilization.

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.
Following QC and counting, proceed immediately to the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression assay.
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