Pseudo-Seq
Method for Genome-Wide Identification of Pseudouridylation Sites
Pseudo-Seq detects pseudouridylation sites in ncRNAs with single-nucleotide resolution using high-throughput sequencing (Carlile et al., 2014). Pseudo-Seq is very similar to PSI-seq, in that both methods use CMC to modify pseudouridines selectively and halt reverse transcription. However, Pseudo-Seq circularizes cDNA strands before PCR amplification and purification, instead of using ARTseq.
Briefly, poly(A)-selected RNA is fragmented and treated with CMC. The RNA is dephosphorylated with CIP and PNK, and size-selected. Next, 3′ adapters are ligated to RNA strands and reverse transcription is initiated. The truncated cDNAs resulting from CMC-modified pseudouridines are purified, circularized, and PCR-amplified. The purified cDNA libraries are sequenced by an NGS method.
Similar methods: PSI-seq, _-Seq, CeU-Seq
Advantages:
- Identifies pseudouridylation sites in ncRNAs
- Provides single-nucleotide resolution
- Identifies peaks by computationally calculating the ratio of reads at the initial mapped position to the total number of reads covering that position (Zaringhalam et al., 2016)
Disadvantages:
- Circularization step may introduce additional bias
- Not yet adopted widely by the scientific community
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Zaringhalam M. and Papavasiliou F. N. Pseudouridylation meets next-generation sequencing. Methods. 2016;107:63-72
References:
Carlile T. M., Rojas-Duran M. F., Zinshteyn B., Shin H., Bartoli K. M. and Gilbert W. V. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature. 2014;
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History: Pseudo-Seq
Revision by sbrumpton on 2017-06-21 07:50:22 - Show/Hide
Method for Genome-Wide Identification of Pseudouridylation Sites
Pseudo-Seq detects pseudouridylation sites in ncRNAs with single-nucleotide resolution using high-throughput sequencing (Carlile et al., 2014). Pseudo-Seq is very similar to PSI-seq, in that both methods use CMC to modify pseudouridines selectively and halt reverse transcription. However, Pseudo-Seq circularizes cDNA strands before PCR amplification and purification, instead of using ARTseq.
Briefly, poly(A)-selected RNA is fragmented and treated with CMC. The RNA is dephosphorylated with CIP and PNK, and size-selected. Next, 3' adapters are ligated to RNA strands and reverse transcription is initiated. The truncated cDNAs resulting from CMC-modified pseudouridines are purified, circularized, and PCR-amplified. The purified cDNA libraries are sequenced by an NGS method.
Similar methods: PSI-seq, _-Seq, CeU-Seq
Advantages:- Identifies pseudouridylation sites in ncRNAs
- Provides single-nucleotide resolution
- Identifies peaks by computationally calculating the ratio of reads at the initial mapped position to the total number of reads covering that position (Zaringhalam et al., 2016)
Disadvantages:- Circularization step may introduce additional bias
- Not yet adopted widely by the scientific community
Reagents:Illumina Library prep and Array Kit SelectorReviews:Zaringhalam M. and Papavasiliou F. N. Pseudouridylation meets next-generation sequencing. Methods. 2016;107:63-72References:Carlile T. M., Rojas-Duran M. F., Zinshteyn B., Shin H., Bartoli K. M. and Gilbert W. V. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature. 2014;