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Full-length mRNA Sequencing

mRNA Sequencing

Sangwon Lee, Magnolia Bostick, Andrew Farmer
R&D, Clontech Laboratories, Inc., a Takara Bio Company
0.0
2

(...) Published on Rev B

  • supported ifcs:
    Open App IFC, mRNA Seq IFC
  • number of ifc runs:
    10

Overview

The SMART-Seq v4 script, which employs a new chemistry (available in the SMART-Seq v4 Ultra Low Input RNA Kit for the Fluidigm C1 System) allows high-quality cDNA synthesis from single cells. Employing locked nucleic acid (LNA) technology integrated with SMART technology, this chemistry offers remarkable sensitivity, enriches for full-length transcripts, and maintains representation of the original mRNA transcripts-critical factors for transcriptome analysis. This chemistry has higher sensitivity than the previous generation (the SMARTer Ultra Low Input RNA Kit for the Fluidigm C1 System that is used in the mRNA Seq script), and detects more genes. In addition, genes with high GC content show higher expression with this chemistry as compared to the previous generation. cDNA libraries generated using this script have been tested for compatibility with Illumina sequencing platforms. User manual is available at www.clontech.com/SMART-Seq-for-Fluidigm. This script has been updated to be compatible with all versions of the C1 Open App IFCs, including the redesigned medium 96 cell IFC.

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Protocol: SMART-Seq v4Duration (H:M): 10:00

Cell Load and Stain

Sample Prep

view protocol worksheet

Tested Primary Cells or Cell Lines

Cell Name Cell Type Source
K562 Cell line
NALM16 Cell line
HELA- FLIP cells Cell line
Jurkat Cell line
GliNS2 Cell line
CB660 Cell line

Performance

The SMART-Seq v4 script delivers high quality, robust and reproducible transcriptomic data. SMART-Seq v4 includes improvements in sensitivity and reduced technical variability relative to the previous mRNA Seq script and the SMARTer Ultra Low Input RNA Kit for the Fluidigm C1 System. Specifically, the SMART-Seq v4 script provides: (1) Higher sensitivity, due to the new chemistry, which produces more cDNA and detects significantly more genes. SMART-Seq v4 achieved a significantly higher exon to intron mapped ratio relative to the previous mRNA Seq script employing the older system. (2) Improved detection of GC-rich genes: more genes with high GC content were identified from the cDNA libraries constructed with SMART-Seq v4 relative to the previous system. (3) Reduced technical variability, with more consistent transcriptomic data among the replicates generated by SMART-Seq v4. This increases the opportunity to detect biological variabilities. See figures 2-5 of the attached poster presented at the NIH 4th Annual Single Cell Analysis Investigators Meeting for performance details.

Publications or Articles

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Performance data

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