Setting Up a Single-Cell Genomic Laboratory
Transcriptomics has been revolutionized by massive throughput RNA-seq. To date, the ongoing decrease in sequencing cost and recent eruption of single-cell related protocols have boosted a demand for single-cell RNA sequencing projects. Although the single
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Introduction Despite the growing interest for single-cell RNA sequencing in the scientific community, only few centers worldwide have the specialized skills and equipment to accommodate the demand for it. Single-cell RNA sequencing (scRNA-Seq) procedures are not standard and are quite challenging for individual laboratories to perform, and not many of them have an open access to specialized facilities [1]. Due to technical limitations, most of the recent research employed “population-level” techniques that should be modified to accommodate new requirements for the single-cell laboratory setup, such as sample and reagent handling, conducting experiments, and QC parameters. One of the factors that should be taken into consideration is that the handling of an individual cell is much more challenging than that of a pool of cells [2]. The minute amount of starting RNA from a single cell is prone to degradation, sample loss, and elevated background noise in sequencing data. To avoid RNA degradation samples should be always kept in a RNase free environment on ice or any other available cooler racks during preparation and reaction setup.
Valentina Proserpio (ed.), Single Cell Methods: Sequencing and Proteomics, Methods in Molecular Biology, vol. 1979, https://doi.org/10.1007/978-1-4939-9240-9_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Lira Mamanova
Fig. 1 PCR workstation/PCR hood
Since a very low amount of RNA is used, extra effort should be taken to avoid sample and reagent contamination. The sources of contamination can be of different nature, including untreated surfaces, pipettes, gloves, equipment, and reagents [3]. An efficient and reliable decontamination procedure should be applied before and after the experiment. RNA-related work should be carried out in a specially designated clean room or in PCR workstation/PCR hood that is equipped with UV lights for sterilization (Fig. 1). In addition, RNase-free reagents, barrier tips, and ultrapure water should be used for scRNA-seq experiment setup [4]. Today’s single-cell studies are typically conducted with thousands of cells per experiment. Most of the standard laboratories employ manual liquid handling, resulting in a quite low throughput, that can be error prone, technically variable, and time-consuming. Automation of scRNA-seq protocols on robotic platforms allows for parallel processing of individual cells at an unprecedented scale and facilitates high-throughput single-cell profiling [5]. Over the past few years, the advances in benchtop automated workstations and dispensing instruments have resulted in increased throughput, accuracy, and reproducibility of scRNA-seq methods (Figs. 2 and 3). It also provides significant reduction in sample reaction volume, minimizes reagent dead volume, tip consumption, and hands-on time that overall results in cost-effective largescale single-cell studies. Another important aspect of automation exploitation is a growing concern regarding the development of musculoskeletal disorders (MSD) that
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