Smart-Seq2
Switch Mechanism at the 5′ End of RNA Templates
Smart-Seq was developed as a single-cell sequencing protocol with improved read coverage across transcripts (Ramskold et al., 2012). Complete coverage across the genome allows the detection of alternative transcript isoforms and SNPs. There are 2 versions of Smart-Seq: Smart-Seq and Smart-seq2. Smart-seq2 includes several improvements over the original Smart-Seq protocol (Picelli et al., 2013) (Picelli et al., 2014). The new protocol includes a locked nucleic acid (LNA), an increased MgCl2 concentration, betaine, and elimination of the purification step to improve the yield significantly.
Smart-seq2: Single cells are lysed in a buffer that contains free dNTPs and oligo(dT)-tailed oligonucleotides with a universal 5′-anchor sequence. RT is performed, which adds 2_5 untemplated nucleotides to the cDNA 3′ end. A template-switching oligo (TSO) is added, carrying 2 riboguanosines and a modified guanosine to produce a LNA as the last base at the 3_ end. After the first-strand reaction, the cDNA is amplified using a limited number of cycles. Next, tagmentation is used to construct sequencing libraries quickly and efficiently from the amplified cDNA.
Advantages:
- As little as 50 pg of starting material can be used
- mRNA sequence does not have to be known
- Improved coverage across transcripts
- High level of mappable reads
Disadvantages:
- Not strand-specific
- No early multiplexing (Shapiro et al., 2013)
- Transcript length bias, with inefficient transcription of reads over 4 Kb (Bhargava et al., 2014)
- Preferential amplification of high-abundance transcripts
- Purification step may lead to loss of material
- Could be subject to strand-invasion bias (Tang et al., 2013)
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
This method has been widely integrated into various sequencing techniques due to its high versatility.
References:
This method has been widely integrated into various sequencing techniques due to its high versatility.
Related
History: Smart-Seq2
Revision by sbrumpton on 2017-06-21 07:50:22 - Show/Hide
Switch Mechanism at the 5' End of RNA Templates
Smart-Seq was developed as a single-cell sequencing protocol with improved read coverage across transcripts (Ramskold et al., 2012). Complete coverage across the genome allows the detection of alternative transcript isoforms and SNPs. There are 2 versions of Smart-Seq: Smart-Seq and Smart-seq2. Smart-seq2 includes several improvements over the original Smart-Seq protocol (Picelli et al., 2013) (Picelli et al., 2014). The new protocol includes a locked nucleic acid (LNA), an increased MgCl2 concentration, betaine, and elimination of the purification step to improve the yield significantly.
Smart-seq2: Single cells are lysed in a buffer that contains free dNTPs and oligo(dT)-tailed oligonucleotides with a universal 5'-anchor sequence. RT is performed, which adds 2_5 untemplated nucleotides to the cDNA 3' end. A template-switching oligo (TSO) is added, carrying 2 riboguanosines and a modified guanosine to produce a LNA as the last base at the 3_ end. After the first-strand reaction, the cDNA is amplified using a limited number of cycles. Next, tagmentation is used to construct sequencing libraries quickly and efficiently from the amplified cDNA.
Advantages:- As little as 50 pg of starting material can be used
- mRNA sequence does not have to be known
- Improved coverage across transcripts
- High level of mappable reads
Disadvantages:- Not strand-specific
- No early multiplexing (Shapiro et al., 2013)
- Transcript length bias, with inefficient transcription of reads over 4 Kb (Bhargava et al., 2014)
- Preferential amplification of high-abundance transcripts
- Purification step may lead to loss of material
- Could be subject to strand-invasion bias (Tang et al., 2013)
Reagents:Illumina Library prep and Array Kit SelectorReviews:This method has been widely integrated into various sequencing techniques due to its high versatility.
References:This method has been widely integrated into various sequencing techniques due to its high versatility.