Single Cell mRNA Sequencing
We provide services for high-throughput single-cell 3´-mRNA sequencing using the nanodroplet-based 10x Genomics platform. The system allows flexible cell capture of 500 – 10,000 single cells per sample, with approximately 60% capture efficiency. The resulting sequencing libraries are compatible with Illumina sequencers.
Description of the Technique
- Single cells are encapsulated into nanoliter-scale droplets together with master mix and gel beads, each bead containing a distinct set of barcoded primers.
- Cell lysis and reverse transcription take place within the droplets, referred to as GEMs (Gel Bead in Emulsion). All cDNA molecules generated within one GEM will share a common barcode sequence.
- After RT-PCR, the barcoded cDNAs from the individual cells are pooled for further amplification and library construction.
- The resulting single-cell libraries are sequenced using the Illumina NovaSeq platform at FFGC. Sequencing data is processed using the CellRanger pipeline, resulting in fastq files.
- Additional data analysis services are available at the Medical Bioinformatics Centre.
Cells must be in a single-cell suspension free of aggregates or debris. It is recommended that cells are passed through a 40 μm strainer.
Accurate cell counts are crucial for reaching targeted number of captured cells. For precise results, it is recommended that cells are counted in four replicates from two independent draws from the cell suspension.
At least 3 x number of cells targeted for analysis must be provided in a concentration of 700-1200 cells per μl.
For example, if 10,000 cells are to be captured, 30,000 cells must be provided.
No data can be recorded from dead or apoptotic cells. We strongly recommend that at least 70% of the cells are viable.
We recommend adhering to sample preparation protocols provided by 10x Genomics.