Single-Cell RNA-Seq by Multiple Annealing and Tailing-Based Quantitative Single-Cell RNA-Seq (MATQ-Seq)
Single-cell technologies have emerged as advanced tools to study various biological processes that demand the single cell resolution. To detect subtle heterogeneity in the transcriptome, high accuracy and sensitivity are still desired for single-cell RNA-
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Introduction The development of new single-cell chemistry and the rapid drop of sequencing cost have greatly propelled the wide applications of single-cell RNA-seq. Single-cell transcriptome profiling has been applied to study embryo development [1] and tissue developmental hierarchy [2, 3], identify new cell types and markers [4, 5], characterize the heterogeneity in tissue and tumor [6], and determine the response of single cells to perturbation [7, 8]. These studies revealed amazing complexity in a variety of biological processes and have revolutionized our view on life science. Multiple methods to profile single-cell transcriptome have been developed in the field [9–17]. Most of these methods utilize oligo dT primer that binds to the PolyA tail of the 30 end of mRNA transcripts for first-strand cDNA synthesis. Three approaches are mostly used for second-strand synthesis. Cel-seq [15, 16] utilizes RNase H to fragment the RNA and uses the fragmented RNA as the primer for second-strand synthesis. SMART-seq [9, 10] takes advantage of terminal transferase activity of MMLV reverse transcriptase which predominantly adds a few bases of C on the 50 end of first-strand cDNA. Template switching oligos are used to bind to
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_5, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Kuanwei Sheng and Chenghang Zong
the C’s and allow for template switching to generate full amplicon for PCR amplification. Tailing-based methods [11] apply PolyA tailing on the 30 of cDNA catalyzed by TdT terminal transferase. Another oligo dT primers are then used to bind to the cDNA PolyA tail to initiate the second-strand synthesis. However, despite the significant progress made in recent years, previous methods are still limited by several factors. Firstly, the capture efficiency of these methods is only 40–50%, meaning that there are at least 50% of transcripts that cannot be detected in a single cell. Secondly, these methods showed strong 30 bias, as secondary structures in the transcripts can easily form and block the elongation of reverse transcription. Thirdly, due to the limitation of the first-strand synthesis, only PolyA transcripts can be characterized. Therefore, these assays are not suitable for studies that demand high sensitivity and accuracy. They are also limited in investigating nonpolyadenylated long noncoding RNA or nascent transcripts. Taking all these into consideration, to quantify subtle transcription variations among single cells, a more quantitative and sensitive single-cell transcriptome assay is needed. We developed a new single-cell RNA-seq assay, multiple annealing and dC-tailing-based quantitative single-cell RNA-seq (MATQ-seq) [18] (Fig. 1). Briefly, after cell lysis, we utilize not only oligo dT but also MALBAC [19] random primers to perform first-strand synthesis. Multiple times of thermocycling is used to allow primers to anneal
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