title: A cost effective 5΄ selective single cell transcriptome profiling approach with improved UMI design.
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.
authors.affiliation: Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, F06560 Sophia Antipolis, France.
authors.forename: Marie-Jeanne
authors.initials: MJ
authors.lastname: Arguel
authors.affiliation: Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, F06560 Sophia Antipolis, France.
authors.forename: Kevin
authors.initials: K
authors.lastname: LeBrigand
authors.affiliation: Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, F06560 Sophia Antipolis, France.
authors.forename: Agnès
authors.initials: A
authors.lastname: Paquet
authors.affiliation: Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, F06560 Sophia Antipolis, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Ruiz García
authors.affiliation: Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, F06560 Sophia Antipolis, France.
authors.forename: Laure-Emmanuelle
authors.initials: LE
authors.lastname: Zaragosi
authors.affiliation: Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, F06560 Sophia Antipolis, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Barbry
authors.affiliation: Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, F06560 Sophia Antipolis, France.
authors.forename: Rainer
authors.initials: R
authors.lastname: Waldmann
mesh_terms: D002478:Cells, Cultured; D018076:DNA, Complementary; D020869:Gene Expression Profiling / Q000191:economics / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D017423:Sequence Analysis, RNA / Q000191:economics / Q000379:methods*; D059010:Single-Cell Analysis
chemical_list: D018076:DNA, Complementary