Smart-Seq
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-Seq: Cells are lysed, and the RNA is hybridized to an oligo(dT)-containing primer. The first strand of the cDNA is synthesized with the addition of a few untemplated C nucleotides. This poly(C) overhang is added exclusively to full-length transcripts. An oligonucleotide primer is hybridized to the poly(C) overhang and used to synthesize the second strand. Full-length cDNAs are PCR-amplified to obtain nanogram amounts of DNA. The PCR products are purified for sequencing.
Advantages:
- mRNA sequence does not have to be known
- As little as 50 pg of starting material can be used
- Improved coverage across transcripts
- High level of mappable reads
Disadvantages:
- Not strand-specific
- No early multiplexing
- Applicable only to poly(A)+ RNA
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-Seq
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-Seq: Cells are lysed, and the RNA is hybridized to an oligo(dT)-containing primer. The first strand of the cDNA is synthesized with the addition of a few untemplated C nucleotides. This poly(C) overhang is added exclusively to full-length transcripts. An oligonucleotide primer is hybridized to the poly(C) overhang and used to synthesize the second strand. Full-length cDNAs are PCR-amplified to obtain nanogram amounts of DNA. The PCR products are purified for sequencing.
Advantages:- mRNA sequence does not have to be known
- As little as 50 pg of starting material can be used
- Improved coverage across transcripts
- High level of mappable reads
Disadvantages:- Not strand-specific
- No early multiplexing
- Applicable only to poly(A)+ RNA
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.