Single-Cell Tagged Reverse Transcription (STRT-Seq)
Single-cell RNA sequencing (scRNA-seq) has become an established approach to profile entire transcriptomes of individual cells from different cell types, tissues, species, and organisms. Single-cell tagged reverse transcription sequencing (STRT-seq) is on
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Introduction Single-cell RNA sequencing (scRNA-seq) is a powerful and unbiased approach for quantifying the transcriptome of individual cells and has transformed our understanding of development and disease mechanism [1]. scRNA-seq approaches has been applied to identify and distinguish subpopulation structures across different cell types including pluripotent stem cell (PSCs), immune cells (Reviewed in [1, 2]) and for all cell types in human body [3, 4]. Single-cell tagged reverse transcription sequencing (STRT or STRT-seq) is one of the early multiplexed approaches for single-cell RNA-sequencing and has been developed to be performed either in 96/384-well plates or using Fluidigm C1 microfluidics system [5, 6]. STRT-seq has further been improved including a recently published dual-index method compatible with nuclear RNA-seq on microwell platform [7, 8]. The initial step in STRT-seq isolation/capture and lysis either in tubes. Subsequently, the first-strand is synthesized using
Electronic supplementary material: The online version of this chapter (https://doi.org/10.1007/978-1-49399240-9_9) contains supplementary material, which is available to authorized users. 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_9, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Kedar Nath Natarajan
biotinylated Oligo-dT primer and the reverse transcriptase adds 3–6 cytosines to the 30 end. Subsequently, using a helper template switching oligonucleotide (TSO), the reverse transcriptase switches the template, continues the helper oligonucleotide synthesis, and introduces a barcode into cDNA. This barcoded cDNA is purified, PCR-amplified, immobilized on biotin beads followed by fragmentation, adapter ligation, library amplification, and sequencing using two sets of primers. The STRT-seq protocol has been improved to incorporate individual molecular counting using unique molecular identifiers (UMI) and adapted to be performed on the Fluidigm C1 platform [5, 7–9]. Herein, I describe the 50 tag counting STRT-seq protocol on 96 single mouse PSCs performed on microfluidics Fluidigm C1 platform (STRT-C1) [6]. The STRT-C1 has several advantages over older STRT-seq protocol and other tag counting scRNA-seq methods. Firstly, STRT-C1 is performed on microfluidics chip that enables accurate, sensitive, and efficient reactions in nanoliter volumes. Secondly, the introduction of UMIs allows for precise counting of unique transcripts and separate from PCR duplicates. Thirdly, the protocol allows flexibility to use in-house Tn5 transposase for tagmentation and different single-cell barcoding indices. Lastly, since the TSO and Oligo-dT have biotin tags, the pooled library can be purified by streptavidin beads and further enriched for 50 UMI transcripts by depleting PvuI restriction site containing 30 fragments. Among the disadvantages of STRT-C1 are costs associated with microfluidics instrument and chips
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