Mar 17, 2025
Human trigeminal samples use Chromium Next GEM Single Cell Multiome protocol
- 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 @ UPennTech. support email: haowu2@upenn.edu
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 21, 2025
Last Modified: March 17, 2025
Protocol Integer ID: 123165
Keywords: Human trigeminal, Chromium Next GEM Single Cell Multiome
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 |
Nuclei isolation
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.On 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. On ice
The tissue homogenate is then passed through a 30mmMACS SmartStrainerfilter (130-098-458) and diluted 1:1 with homogenization buffer.On 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).4 °C
Library preparation
Library preparation
For 10x multiome sequencing analysis of Human trigeminal, we followed the standard library preparation protocol from 10x Genomics (see below).
CG000338_ChromiumNextGEM_Multiome_ATAC_GEX_User_Guide_RevG.pdf.pdf4.7MB Protocol references
- Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing. https://www.cell.com/neuron/pdf/S0896-6273(22)00228-8.pdf
- 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