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Nucleic Acids Res. 2017 Apr 20;45(7):e48. doi: 10.1093/nar/gkw1242.

A cost effective 5΄ selective single cell transcriptome profiling approach with improved UMI design.

Author information

1
Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, F06560 Sophia Antipolis, France.

Abstract

Single cell RNA sequencing approaches are instrumental in studies of cell-to-cell variability. 5΄ selective transcriptome profiling approaches allow simultaneous definition of the transcription start size and have advantages over 3΄ selective approaches which just provide internal sequences close to the 3΄ end. The only currently existing 5΄ selective approach requires costly and labor intensive fragmentation and cell barcoding after cDNA amplification. We developed an optimized 5΄ selective workflow where all the cell indexing is done prior to fragmentation. With our protocol, cell indexing can be performed in the Fluidigm C1 microfluidic device, resulting in a significant reduction of cost and labor. We also designed optimized unique molecular identifiers that show less sequence bias and vulnerability towards sequencing errors resulting in an improved accuracy of molecule counting. We provide comprehensive experimental workflows for Illumina and Ion Proton sequencers that allow single cell sequencing in a cost range comparable to qPCR assays.

PMID:
27940562
PMCID:
PMC5397152
DOI:
10.1093/nar/gkw1242
[Indexed for MEDLINE]
Free PMC Article

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