Mar 17, 2025

Public workspaceHuman trigeminal samples use Chromium Next GEM Single Cell Multiome protocol 

DOI
https://dx.doi.org/10.17504/protocols.io.3byl4wo4ovo5/v1
  • Dongming Liang1,2,
  • Hao Wu1,2
  • 1Department of Genetics, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia PA 19104, USA;
  • 2The Institute for Regenerative Medicine, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
  • Wu Lab @ UPenn
  • PRECISION Human Pain Network
Dongming Liang
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl4wo4ovo5/v1
Protocol Citation: Dongming Liang, Hao Wu 2025. Human trigeminal samples use Chromium Next GEM Single Cell Multiome protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wo4ovo5/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 20, 2025
Last Modified: March 17, 2025
Protocol Integer ID: 123165
Keywords: Human trigeminal, Chromium Next GEM Single Cell Multiome, chromium next gem single cell multiome protocol, next gem single cell multiome protocol, 10x genomics multiome assay, simultaneous profiling of gene expression, chromatin, accessible chromatin, linking open chromatin region, chromatin within the same cell, cells with similar transcriptional profile, human trigeminal sample, open chromatin region, distinct chromatin landscape, drivers of differential gene expression, differential gene expression, similar transcriptional profile, deeper mechanistic insights into dynamic gene, gene expression, single cell, comprehensive view of cellular regulation, cellular regulation, thousands of cell, rna, multiomic approach, integrating rna, same cell, dynamic gene
Abstract
The 10x Genomics Multiome assay enables simultaneous profiling of gene expression and open chromatin within the same cell across thousands of cells (ref 1-2), offering a comprehensive view of cellular regulation. By integrating RNA sequencing with assay for transposase-accessible chromatin (ATAC), this multiomic approach enhances the resolution of cell states, identifies drivers of differential gene expression, and reveals cells with similar transcriptional profiles but distinct chromatin landscapes. By linking open chromatin regions to gene expression at single-cell resolution, this technology could provide deeper mechanistic insights into dynamic gene regulatory networks of Human trigeminal normal and migraine
Materials
Reagents Vol. μL 5ml final concentration
5 M NaCl 14.6 73 73 mM NaCl
0.5 M CaCl2 1 5 0.5mM CaCl2
1 M MgCl2 10 50 10mM MgCl2
1 MTris-HCl pH 7.5 5 25 5mMTris-HCl pH 7.5
5%BSA 200 1000 1%BSA
40 U/ul Protector RNase inhibitor 2.5 12.5 0.1U/ul Protector RNase inhibitor
H2O 766.9 3834.5
1000 5000
10% IGPAL 20 0.2% IGPAL
Troubleshooting
Nuclei isolation
Preparation of Single Trigeminal Nuclei
Place 100um thin tissue sections Human TG from OCT-embedded sample in a 1.5 mL Eppendorf tube.

Place thin tissue sections Human TG ganglia in homogenization buffer [73mMNaCl, 0.5mM CaCl2, 10mMMgCl2,5mMTris-HCl pH7.5, 1%BSA, 0.1U/ul Protector RNase inhibitor (Millipore Sigma 3335399001)] for 15 s on ice using chilled reagents.TemperatureOn ice

Transfer samples to a prechilled Douncehomogenizer in 5mL homogenization bufferfor 10 strokes with a loose pestle, then add IGPAL (Millipore Sigma 542334) to a final concentration of 0.2% and apply 5 ADDITIONAL strokes with a tight pestle. TemperatureOn ice

The tissue homogenate is then passed through a 30mmMACS SmartStrainerfilter (130-098-458) and diluted 1:1 with homogenization buffer.TemperatureOn ice

Nuclei are centrifugedat 500 g for 10 min at 4°Cand resuspended in 1ml of Resuspension buffer (1X PBS, 0.04%BSA, and0.1U/mlRNase inhibitor).Temperature4 °C

Library preparation
For 10x multiome sequencing analysis of Human trigeminal, we followed the standard library preparation protocol from 10x Genomics (see below).
Download CG000338_ChromiumNextGEM_Multiome_ATAC_GEX_User_Guide_RevG.pdf.pdfCG000338_ChromiumNextGEM_Multiome_ATAC_GEX_User_Guide_RevG.pdf.pdf4.7MB

Protocol references
  1. Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing. https://www.cell.com/neuron/pdf/S0896-6273(22)00228-8.pdf
  2. Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits User Guide. https://www.10xgenomics.com/support/epi-multiome/documentation/steps/library-prep/chromium-next-gem-single-cell-multiome-atac-plus-gene-expression-reagent-kits-user-guide