Single-cell full-length transcriptome sequencing refers to the use of PacBio SMRT or Nanopore sequencing technology to directly read the reverse-transcribed full-length cDNA of single cells treated with 10X Genomics technologies, identifying and quantifying the Isoform of each cell by analysis, and detecting alternative splicing events at the single-cell level. Single-cell full-length transcriptome sequencing improves single-cell transcriptome analysis from the gene level to the isoform level by combining the advantages of single cells with three-generation long-read sequencing technology.
To date, multiple studies have demonstrated extensive Isoform diversity among different cell types. Single-Cell Full-Length RNA Sequencing combines the advantages of scRNA-seq and Iso-Seq technology to realize the study of full-length transcripts at single-cell level and analyze single-cell gene expression. Compared with ordinary transcriptome sequencing, Single-Cell Full-Length RNA Sequencing obtains variable splicing information through short-read splicing, and Iso-Seq directly obtains full-length transcript sequences, which opens the way for accurate variable splicing research. However, at present, Iso-Seq is mainly used for Isoform analysis of the whole tissue, and its successful application on single cells is limited to a small number of cells. Single-Cell Full-Length RNA Sequencing utilizes the 10x Genomics single-cell platform to capture thousands to tens of thousands of single cells and amplify full-length cDNA. The above cDNA was then divided into two groups, one for 10x Genomics single-cell transcriptome library construction and sequencing, the other for the same cell-specific 10x Barcode-labeled full-length cDNA. CD Genomics uses self-optimized library construction technology based on PacBio SMRT or Nanopore library to construct full-length transcriptome library, and perform sequencing on PacBio SMRT or Nanopore platform. Finally, a joint analysis of the above data can be achieved to determine the single-cell origin of each full-length transcriptome sequence, thereby enabling Isoform's cell type classification.
|This method can be applied to any species, from microorganisms to humans.
|Quantitative analysis down to single-cell levels for input samples.
|Discovery of more cellular differences based on high resolution analysis.
|Understanding complex tissues, tumor heterogeneity and clonal evolution.
Isolation of viable, single cells from a given sample
Total RNA or poly-A RNA; Smart-seq2
10X Genomics Chromium X;
Nanopore ONT Platforms;
PacBio SR II and PacBio Sequel
Visualize and preprocess results, and perform custom bioinformatics analysis.
RNA amount: Total RNA ≥ 5 ug (without degradation or DNA contamination); RNA purity: OD260/280 = 1.8~2.2; OD260/230 ≥ 1.5; RNA quality: 28S:18S ≥ 1.5，RIN ≥ 7
Please make sure that the RNA is not significantly degraded.
Sample storage: RNA can be dissolved in ethanol or RNA-free ultra-pure water and stored at -80°C. RNA should avoid repeated freezing and thawing.
Shipping Method: When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.
Deliverable: FastQ, BAM, coverage summary, QC report, custom bioinformatics analysis.