The Smart-seq2 method exploits the so-called SMART reaction (Switching Mechanism At the end of the 5′-end of the RNA Transcript), developed by scientists at CLONTECH several years ago (Chenchik et al., 1996; Zhu et al., 2001). In 2010 Clontech was the first company to commercialize a kit for ultra-low RNA input called SMARTer® Ultra® Low RNA kit (now on the v4 release; Patent No. JP4043516 and EP0871780). This method is nowadays simply known as “Smart-seq”.
Smart-seq2 represents the second generation of this method and was developed in Rickard Sandberg’s lab at Karolinska Institute in 2013 (Picelli et al., 2013, Picelli et al., 2014). Smart-seq2 exploits two intrinsic properties of the Moloney Murine Leukemia Virus (MMLV) reverse transcriptase: Reverse Transcription (RT) and Template Switching (TS). Cells are lysed in a mild hypotonic buffer containing Triton X-100 and a RNAse inhibitor to prevent RNA degradation. The poly-adenylated cytoplasmic mRNA is then primed with an anchored oligo-dT primer carrying a known sequence at the 5′- end and the RT is performed until the enzyme has reached the 5′-end of each mRNA transcript (Figure 1).
Template switching is the ability of the MMLV reverse transcriptase to introduce a few untemplated nucleotides, predominantly 2-5 cytosines, when it reaches the 5′-end of the RNA template, corresponding to the 3′-end of the newly synthesized cDNA strand. These extra nucleotides work as a docking site for a helper oligonucleotide (“Template Switching Oligonucleotide”, or TSO) that, in the first Smart-seq kit, carried 3 riboguanosines at its 3′-end. The reverse transcriptase is then able to “switch template” (from mRNA to the DNA of the TSO) and synthesize a complementary DNA strand using the helper oligonucleotide as template. Thus, TS makes possible the introduction of an arbitrary sequence at the end of the transcript and, along with the known sequence located at the 5′-end of the oligo-dT primer, allows the efficient amplification of all the transcripts in a cell in the following PCR step. Because the sequence on both sides of each cDNA is the same the PCR can be carried out with just one primer, thus exploiting the PCR suppression effect.
The TSO in the Smart-seq2 method replaces the terminal riboguanosine with a locked nucleic acid (LNA)-modified deoxyguanosine (Figure 1). Locked nucleotides are characterized by an internal bond between the O2′ and the C4′ of the furanose ring, linked by a methylene group. The modification introduces a conformational lock in the molecule, which nonetheless still retains the physical properties of the native nucleic acid. Two interesting properties of LNAs are advantageous for this application: the enhanced thermal stability of the LNA monomers and their ability to anneal strongly to the untemplated 3′ extension of the cDNA.
Although the original Smart-seq method dramatically represented an improvement in terms transcriptome coverage and and sensitivity compared to previous methods, it bore some important limitations. In particular, there was a lower read coverage towards the 5′-end compared to the 3′-end of the transcripts, especially for those spanning several kilobases. Moreover, in the final sequencing library, an under-representation of transcripts with a high GC-content was observed, presumably an effect of the complex secondary structure of the RNA that the DNA polymerase could not overcome during the PCR.
Smart-seq2 addresses all these limitations by making use of different additives and an innovative DNA polymerase for amplifying the reverse-transcribed cDNA (Picelli et al., 2013). An additional advantage of Smart-seq2 is that it entirely relies on off-the-shelf reagents, thus making it more affordable for researchers on a tight budget.
After PCR the double-stranded cDNA is used for the generation of sequencing-ready libraries by using the Nextera XT DNA Library Preparation kit (Illumina). Library preparation represents the largest cost item of the entire protocol, in particular for full-length methods where the early indexing is not feasible and in which separate reactions need to be performed for each sample.
The Nextera XT kit relies on a hyperactive variant of a Tn5 transposase that carries out the fragmentation of double-stranded DNA and ligates synthetic oligonucleotides (“tags”) at both ends in a 5-minute reaction (Adey et al., 2010). Since the DNA is simultaneously tagged and fragmented, the reaction has been named “tagmentation”. A second PCR is then needed to append barcode adaptors for multiplexing (Figure 1). Although the commercial Nextera XT kit is extremely robust and versatile, the price represents a problem when thousands of single cells need to be analyzed or when the financial resources are limited.
A (partial) solution to the problem that we devised at the ESCG Facility is a 10-times reduction of reaction volumes. Other groups have also shown that the volume of the tagmentation reaction can be reduced by 100-fold using acoustic droplet ejection without negatively affecting the quality of the final data (Shapland et al., 2015).
Smart-seq2 still has some important limitations and it is important to keep them in mind when deciding which RNA-sequencing method is the most suitable for answering a specific biological question.
- The information about strand-specificity is lost in the PCR step unless the long full-length transcripts are sequenced by using the Single Molecule Real-Time (SMRT) Sequencing technology developed by Pacific Biosciences which has an average read length of > 10 kb. At the ESCG Facility we do NOT offer such an option but rather performs dual-end 50 bp sequencing using Illumina HiSeq 2500 instruments.
- Being a full-length RNA-sequencing method, Smart-seq2 does not allow the introduction of Unique Molecular Identifiers (UMIs) for molecular counting. Since this barcoding would label only the terminal portion of each transcript, the identity of the internal fragments after tagmentation would be indistinguishable between different cells. To partly correct for this issue we introduced the use of artificial RNA spike-ins, such as those developed by the External RNA Controls Consortium (ERCC).
- Samples can be pooled just prior to sequencing, making the method more labor-intensive than tag-based sequencing methods such as STRT-seq.
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