What Are the Principles of Single-Cell Sequencing Using 10x Genomics Technology?
10x Genomics technology is a groundbreaking single-cell sequencing approach that enables the acquisition of genomic, transcriptomic, and epigenomic data at single-cell resolution. The principles of this technology comprise the following key steps:
Preparation of Single-Cell Suspension
Tissue or cell samples are enzymatically or mechanically dissociated into single-cell suspensions.
Microdroplet Encapsulation
The single-cell suspension is mixed with barcoded gel beads, each carrying a unique molecular barcode. Through microfluidic technology, individual cells are co-encapsulated with single gel beads into microdroplets. Ideally, each microdroplet contains only one cell and one bead.
mRNA Capture and Barcode Labeling
Cells are lysed within the droplets, releasing mRNA molecules. These mRNA molecules hybridize to oligonucleotide probes on the gel beads. The probes contain poly-T sequences that bind to the poly-A tails at the 3′ ends of the mRNA. This allows the mRNA to be captured by probes bearing cell-specific barcodes and Unique Molecular Identifiers (UMIs). UMIs mitigate amplification bias and enable more accurate quantification of original mRNA transcripts.
Reverse Transcription and Amplification
The captured mRNA is reverse-transcribed into cDNA and then PCR-amplified within the droplets. During this process, both cell-specific barcodes and UMIs are retained and incorporated into each cDNA molecule.
cDNA Library Preparation
Amplified cDNA is extracted from the droplets and used to construct a sequencing library. This library contains cDNA molecules labeled with both cell-specific barcodes and UMIs.
High-Throughput Sequencing
The cDNA library is sequenced using high-throughput platforms such as Illumina. The resulting data facilitate downstream analyses, including gene expression profiling, assessment of cellular heterogeneity, and identification of distinct cell subpopulations.
Data Analysis
Sequencing reads are processed and assigned to individual cells, and gene expression levels are quantified based on UMI counts.
These data support further bioinformatics analyses, such as clustering of cells, identification of differentially expressed genes, and exploration of cell-cell interactions.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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