Deep sequencing has been revolutionizing biology and medicine in recent years, providing single base-level precision for our understanding of nucleic acid sequences in high throughput fashion. Sequencing of RNA, or RNA-Seq, is now a common method to analyze gene expression and to uncover novel RNA species. Aspects of RNA biogenesis and metabolism can be interrogated with specialized methods for cDNA library preparation. In this study, the authors review current RNA-Seq methods for general analysis of gene expression and several specific applications, including isoform and gene fusion detection, digital gene expression profiling, targeted sequencing and single-cell analysis. In addition, we discuss approaches to examine aspects of RNA in the cell, technical challenges of existing RNA-Seq methods, and future directions.
RNA‐Seq of single cells
(a) Reverse transcription with oligo‐dT primers and a universal primer sequence is followed by poly(A) tailing. After PCR amplifications, standard RNA‐Seq libraries are prepared. (b) Reverse transcription incorporates a universal primer sequence. Template switching of reverse transcription is followed by annealing of the oligonucleotide with the sequence for a second PCR primer. (c) cDNA synthesis introduces the T7 promoter sequence at the 5′ end. After second strand cDNA synthesis, cRNA copies are generated by in vitro transcription. Finally, the second adapter is ligated to the 3′ end of the cRNA and libraries are constructed by PCR amplification. (d) Single‐cell MALBAC RNA‐Seq. Primers with seven random nucleotides at the 3′ end are annealed to cDNA and extended. Amplicons are looped to protect them from being further amplified. Ten cycles of quasilinear amplification are followed by exponential PCR.
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