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Developmental biology - RNA Profiles|
Profiling Each RNA
Single-cell RNA sequencing makes it possible to obtain a snapshot of the functional state of any given cell - a molecular fingerprint, as it were.
Essentially, the new technique determines the composition of messenger RNA (mRNA) in a cell. mRNAs are copies ('transcripts') of defined segments of genetic information encoded in a cell's DNA. They serve as blueprints for the synthesis by specialized organelles called ribosomes of specific proteins required by each cell type.
Thus the inventory of the mRNAs present in a cell amounts to a list of the proteins made by that cell, which essentially reveals its functional state. By identifying the genes that were active at the time of analysis, it can tell us how these genes are regulated, and what happens when this process is disrupted by infection or other disease states.
The sequencing of all mRNAs from a single cell is a demanding task, and several different procedures were designed and implemented. All begin with "reverse transcription" of the isolated mRNAs into DNA by enzymes known as reverse transcriptases.
"The trick is to supplement the reverse transcriptase reaction with an agent that increases the density of the medium. This induces molecular crowding, and speeds up the reaction, so that more RNA molecules are transcribed into DNA strands," Enard explains. A second modification reduces the incidence of preferential amplification of certain DNAs, which would otherwise distort the representation of the different RNAs present in the original cell. He adds: "Together, these modifications make our method, mcSCRB-seq, one of the most effective and economical RNA-seq procedures currently available."
Single-cell RNA sequencing methods are also indispensable for the realization of a Human Cell Atlas. This ambitious international project is comparable in scale to the Human Genome Project. Its goal is to assemble a catalog of all human cell types, from embryo to adult, based on their specific patterns of gene activity. The project promises to vastly expand our knowledge of human biology and the origins of human diseases.
Single-cell RNA sequencing (scRNA-seq) has emerged as a central genome-wide method to characterize cellular identities and processes. Consequently, improving its sensitivity, flexibility, and cost-efficiency can advance many research questions. Among the flexible plate-based methods, single-cell RNA barcoding and sequencing (SCRB-seq) is highly sensitive and efficient. Here, we systematically evaluate experimental conditions of this protocol and find that adding polyethylene glycol considerably increases sensitivity by enhancing cDNA synthesis. Furthermore, using Terra polymerase increases efficiency due to a more even cDNA amplification that requires less sequencing of libraries. We combined these and other improvements to develop a scRNA-seq library protocol we call molecular crowding SCRB-seq (mcSCRB-seq), which we show to be one of the most sensitive, efficient, and flexible scRNA-seq methods to date.
Authors: Johannes W. Bagnoli, Christoph Ziegenhain, Aleksandar Janjic, Lucas E. Wange, Beate Vieth, Swati Parekh, Johanna Geuder, Ines Hellmann and Wolfgang Enard
We thank Ines Bliesener for expert technical assistance. We are grateful to Magali Soumillon and Tarjei Mikkelsen for providing the original SCRB-seq protocol and to Stefan Krebs and Helmut Blum for sequencing. We would like to thank Elena Winheim for the PBMC sample. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through LMUexcellent and the SFB1243 (Subproject A14/A15).
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How The Protocol Works
a. Overview of the mcSCRB-seq protocol workflow. Single cells are isolated via FACS into multiwell plates containing lysis buffer, barcoded oligo-dT primers, and Proteinase K. Reverse transcription and template switching are carried out in the presence of 7.5% PEG 8000 to induce molecular crowding conditions. After pooling the barcoded cDNA with magnetic SPRI beads, PCR amplification using Terra polymerase is performed. Image Credit: Nature Communications