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.

Features

Any Species Single-Cell Insights High Resolution Multiple Applications
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.

Project Workflow

Sample Preparation

1. Sample Preparation

Isolation of viable, single cells from a given sample

Library Preparation

2. Library Preparation

Total RNA or poly-A RNA; Smart-seq2

Sequencing

3. Sequencing Platform

10X Genomics Chromium X;
Nanopore ONT Platforms;
PacBio SR II and PacBio Sequel

Data Analysis

4. Data Analysis

Visualize and preprocess results, and perform custom bioinformatics analysis.

Single-Cell Full-Length RNA Sequencing

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: 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.

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).
* For Research Use Only. Not for use in diagnostic procedures.


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