Single Cell mRNA Sequencing

    We provide services for high-throughput single-cell mRNA sequencing using the nanodroplet-based Chromium™ Controller from 10x Genomics. The resulting sequencing libraries are compatible with Illumina sequencing platforms.

    Description of the Technique

    1. Construction of Single Cell Sequencing Libraries using the 10x Genomics Chromium Controller

    • Encapsulation of the cells

    Single cell are encapsulated with gel beads and master mix into nanoscale droplets. Primers with Unique Molecular Identifier (UMI), or “barcode” are linked to the gel beads.

    • Cell lysis and RT-PCR, take place within the droplets, referred to as GEMs (Gel Bead in Emulsion)
    • After RT-PCR, the barcoded cDNA from single cells is pooled for further amplification and library construction. The libraries are indexed and can be multiplexed if necessary.

     

     

    Final library construct

    Key features :

    • Throughput of up to 10,000 single cells per sample.
    • Theoretical multiplet rate ranges from 0.4% of 500 recovered cells to 7.6% of 10,000 recovered cells.
    • 1-8 samples can be processed in parallel.

    2. Sequencing of the Single Cell Libraries

    The single-cell libraries can be sequenced using the Illumina NovaSeq platform available in-house at FFGC.

    3. Data Analysis with 10x Genomics Software

    Sequencing data is processed and aligned using the CellRanger pipeline from 10x Genomics. The following files are provided to the user:

    • Quality Control summary in html format
    • Gene expression data as a gene-barcode matrix in hd5 format 
    • Raw sequencing data in fastq or bam format (upon request)
    • Loupe analysis package, which can be explored using the freely available Loupe Cell Browser software

    A plethora of tools for scRNA-seq data analysis has been developed. An extensive listing of these can be found at scrna-tools.org.
    Bespoke analysis service is available through the Medical Bioinformatics Centre.

     


    Sample Requirements

    • Samples can be either freshly prepared or viable frozen cells.
    • No data can be recorded from dead or apoptotic cells. Presence of such cells in the sample will decrease the number of cells detected by the analysis and might compromise data quality.
    • The nominal cell capture efficiency is about 60%, but we highly recommend starting with at least 50,000 cells.
    • The protocol is compatible with cell sorting and MACS purification.
    • Cells from solid tissues can be analysed, provided that the dissociation process does not compromise viability.
    • The samples must be in a single-cell suspension, without aggregates, excess debris or contaminants.
    • We recommend adhering to sample preparation protocols provided by 10x Genomics.