10x Chromium Single Cell Multiome ATAC + Gene Expression
The 10x Chromium Single Cell Multiome ATAC + Gene Expression solution allows the simultaneous profiling of accesible chromatin and gene expression from individual cell nuclei ranging from 500 - 10,000 nuclei per sample and up to 80,000 nuclei per run. Single cell multiome interrogation allows the characterization of complex cell populations and the identification of cellular heterogeneity exposing insights from combined epigenomic and gene expression profiling at a single cell level. The dual profiling allowing the discovery of regulatory elements and gene expression interactions that drive cell differentiation, development, and disease.
- Combined detection of mRNA and open chromatin from the same cell
- Discover new gene regulatory interactions
- Detect rare cell populations with single cell precision
Single nuclei multiome libraries prepared using the 10x Chromium X are Illumina compatable and will be sequenced using a NovaSeq 6000. The ATAC and expression libraries require different sequencing strategies. A 100 cycle symetrical sequencing run is required for the ATAC libraries. A 100 cycle asymetrical sequencing strategy is required for the expression libraries. For a typical multiome project targeting 10,000 nuclei @ 25,000 read pairs per nuclei for both the ATAC and expression libraries, 2 - full S1 flow cells will be required to sequence 8 ATAC libraries and 8 expression libraries. 2 - SP full flow cell runs will meet the sequencing demand for a 4 sample single cell multiome project.
The success of a single nuclei multiome sequencing project is dependent on the quality of the cell nuclei preparation provided to the Genomics Core. The core recommends using the Chromium Nuclei Isolation Kit (10x Genomics 1000493) to propare your nuclei isolates and can provide a 10x quote number for discounted pricing. The researcher must coordinate their sample delivery with the core personnel at least 2 weeks prior to the submission of nuclei. The nuclei sample must be submitted as a completely dissociated nuclei suspension derived from cells with a high viability percentage of >80%. Crypreserved cells or tissue can be used for nuclei isolation. For cells undergoing flow cytometry sorting prior to nuclei isolation, additional coordination between the Flow Cytometry Core Laboratory and the Genomics Core will be required. The concentration and viability of each nuclei suspension will be assayed using the Contess II FL Automated Cell Counter. An assessment of nuclei viability, at a high magnification ≥ 50x is ideal to ensure that blebbing of the nuclei is not present or prevalent (magnified images are requested).
- Plan your nuclei preparation time frame to conform with the predetermined Genomics Core submission schedule. Contact the Genomics Core Staff 2 weeks prior to your submission date to coordinate your Multiome project.
- Fresh cells, tissue or cryopreserved cells can be used for nuclei isolation.
- Primary/fragile cells may have high amounts of ambient/background DNA. Treating the cells with DNase I prior to nuclei isolation can remove ambient DNA improving the quality of Single Cell Multiome ATAC libraries. See 10x Demonstrated Protocol : Nuclei Isolation for Single Cell ATAC Sequencing in the links below.
- Solid tissues and other large cell aggregates must be disassociated using mechanical or enzymatic disassociation.
- It is recommended that a trial nuclei isolation procedure be performed before submitting a nuclei prep to the Genomics Core to determine the nuclei concentration and clarity of the suspension.
- Critical : Resuspend nuclei isolates in 1x Nuclei Buffer using nuclease free water as the diluent prior to sample submission. Nuclei Buffer 20x (10x Genomics PN-2000153 / 2000207). Nuclei Buffer is optimized for the transposition and barcoding steps in the Single Cell Multiome ATAC + Expression protocol.
- Minimize delay between nuclei preparation and submission. Keep nuclei on ice.
- To ensure a well singulated nuclei suspension free from cell debris, nuclei straining can be employed. Ensure that pore size is larger than the nuclei diameter but small enough to catch clumps and debris. Account for volume loss associated with the straining process and repeat the nuclei count to correct for loss.
- Submit nuclei samples with a concentration of 3,080 - 7,700 nuclei/ul (3 - 7.7 million nuclei/ml) to target 10,000 nuclei for interrogation.
- An aliquot of 1X Nuclei Buffer can be provided by the Genomics Core personnel when coordinating your Multiome ATAC + Expression project with the core.
Supporting documents and video links for nuclei isolation and project planning
- 10x Video: Single Cell Multiome ATAC plus Gene Expression
- Chromium Nuclei Isolation Kit - User Guide
- Chromium Nuclei Isolation Kit - Tested Tissues
- 10x Video: Chromium Nuclei Isolation - How-To Series
- Demonstrated Protocol: Nuclei Isolation for Single Cell ATAC Sequencing
- Demostrated Protocol: Nuclei Isolation from Complex Tissue for Multiome
- Demonstrated Protocol: Nuclei Isolation From Embryonic Mouse Brain Tissue
- Pipette Tip Recommendations for 10x Genomics Single Cell/Nuclei Protocols
- 10x Genomics Community Forum