Single-Cell Full-Length RNA Sequencing

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.

Overview

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.

Project Workflow

Bioinformatics Analysis Pipeline

In-depth data analysis:

• Single cell level variable shear 
• Full-length Isoform analysis: quantitative isoform analysis, novel Isoforms
• Transcriptome annotation (Gene Ontology, KEGG pathway, KOG or COG, Swissport)
• Gene Structures analysis, including alternative splicing, LncRNA, SSR, CDS
• Gene expression level analysis
• Differentially expressed gene analysis
• Differentially expressed transcriptome KEGG enrichment analysis
• Differentially expressed gene heatmap

Sample Requirements

RNA amount: Total RNA ≥ 5 ug (without degradation or DNA contamination); RNA purity: OD_260/280 = 1.8~2.2; OD_260/230 ≥ 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.

References:

1. Hayashi T, Ozaki H, Sasagawa Y, et al. Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs. Nature communications. 2018 Feb 12;9(1).
2. Kolodziejczyk AA, Kim JK, Svensson V, et al. The technology and biology of single-cell RNA sequencing. Molecular cell. 2015 May 21;58(4).