Single cell transcription profiling provides the opportunity to stratify complex cell types from heterogeneous tissue, to identify rare cell types, and to characterize subpopulations from seemingly homogeneous populations of cells. Given the ability of the technique to identify subtle expression changes at a single cell level, the approach is ideal for developing normative expression in atlas-type studies or assessing the impact that disease or treatment states may have on tissues and cells. The Molecular Genomics Core has been carrying out scRNAseq since 2016. As of September 2023, we have run over 1500 scRNAseq samples from over 100 investigators across 9 different species, giving us confidence in the data we generate.”
Single Cell Gene Expression Flex
10x Genomics’ Flex Assay: Gene expression profiling of single cell/nuclei samples fixed with formaldehyde or isolated from FFPE tissue blocks. The Flex assay allows for use of precious samples that were previously inaccessible due to logistical challenges in sample handling. Flex provides flexibility in the sample preparation process and experimental design by enabling batching across time and collection sites, cell surface protein labelling, sample multiplexing with unique sample barcodes, infectious agent neutralization, and the potential for cost savings. The assay is a probe-based chemistry that requires less sequencing depth than traditional scRNAseq, and covers 18,000 human genes or 19,000 mouse genes, including mitochondrial genes for assessing sample quality. Flex allows for the targeting of as few as 500 cells up to 128k cells with multiplexing and pooling.
Single Nuclei RNAseq (snRNA-seq)
Optimally, single cell RNA sequencing data is generated from freshly dissociated cells with high viability. However, in some circumstances it is challenging to collect fresh biospecimens, the tissue may be a legacy sample from frozen storage, or tissue dissociation may cause lysing of specifically labile cell types. To circumvent these problems, and with development support from the Duke SoM, the MGC has optimized nuclei isolation protocols to generate snRNA-Seq data. We have applied our new protocol to a two-year-old frozen brain tumor sample to assess the use of legacy samples, and fresh mouse brain tissue to capture sensitive neuronal expression profiles, using the 10xGenomics Chromium platform.
For more information, visit 10xGenomics.com.
The tightly packed eukaryotic genome relies on the highly regulated alteration of chromatin structure to facilitate the accessibility of genomic regions for gene expression. These chromatin alterations within the genome can reveal a great deal about the state of the cell. Assay for Transposase-Accessible Chromatin (ATAC-seq) coupled with next-generation sequencing is a technique to identify accessible chromatin regions. The 10x Genomics’ Chromium Single Cell ATAC Sequencing Solution (scATAC-Seq) is capable of identifying rare and important cellular subtypes that cannot be acquired in amounts sufficient for genome-wide chromatin analyses. With as little as a few hundred cells, scATAC-Seq allows profiling of chromatin structure at the single cell level, providing an unprecedented view of the heterogeneity of chromatin regulation.
Single Cell ATAC + Gene Expression
Chromium Single Cell Multiome + Gene Expression unites transcriptional and chromatin landscapes from a single nucleus. This multiomic approach characterizes direct linkages between gene expression and regulatory elements across tens of thousands of cells giving a more comprehensive view of the cell state, differential expression drivers and functional differences within cell populations. This product allows an investigator to leverage two modalities at once, maximizing data collection and minimizing sample depletion.
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The ability to profile immune receptor clonotypes confers the ability to predict the success of stem cell therapy, establish the impact of antigenic exposure, and characterize the properties of tumor infiltrating lymphocytes, amongst other applications. Previous approaches have been limited to heavy chain profiling of specificity conferring regions of the genome, however the 10x Genomics Single Cell V(D)J application offers a more comprehensive solution for measuring immune repertoire information and gene expression from the same single cell. Importantly, paired T-cell receptor (TCR) or B-cell immunoglobulin (Ig) receptor sequences are profiled from 100-10,000 individual cells per sample.
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Single cell expression profiling via scRNA-Seq and snRNA-Seq provides an invaluable snapshot of cell state, but does not predict the translational outcome of mRNA expression. To do this, 10x Genomics have adopted Feature Barcoding technology that uses their microfluidic platform to assess concomitantly single cell gene and protein expression. A pool of ~3,500,000 10x Barcodes separately index each cell’s transcriptome and surface proteins by partitioning between 500 – 10,000 cells using the platforms nanoliter-scale gel bead technology. Protein specific antibodies with custom ligated adapters are bound to the target cells, cells are partitioned into specific GEMs, and proteins and transcripts are assigned to a specific cell via barcodes, and then proteins and mRNA are quantitatively measured using unique molecular indices.
Future Development for Single Cell Feature Barcoding
Single Cell Gene Expression and Crispr Screening
Highlights
- Simultaneously assess perturbation phenotypes and gene expression from the same cell
- Enable high throughput and high resolution functional genetic screens in hundreds to tens of thousands of cells simultaneously
- Determine comprehensive gene expression phenotypes for individual perturbations
- Customize your CRISPR pools with dozens, to hundreds, to thousands of gRNAs
- Implement an improved and novel methodology over published methods by directly capturing and sequencing gRNAs, eliminating the need for a proxy barcode
For more information, visit 10xGenomics.com.
Leverage CellPlex for sample multiplexing to enable flexible experimental design with greater throughput for your single cell gene expression and immune profiling experiments. Combine samples or profile more cells across diverse conditions, including varied primary samples, time-course studies, dose-response titrations, and more, in a single run. Combine multiple samples in one multiplexed library to save on library generation costs.
For more information, visit 10xGenomics.com