PSI-Seq
Pseudouridine Site Identification Sequencing
PSI-Seq identifies RNA sequences containing pseudouridine sites using high-throughput sequencing (Lovejoy et al., 2014). PSI-Seq uses N-Cyclohexyl-N_-(2-morpholinoethyl)carbodiimide (CMC) to modify pseudouridines selectively, effectively halting reverse transcription. The cDNA libraries are prepared by the ARTseq method.
Briefly, samples are poly(A)-selected, treated with DNase, and fragmented. CMC is added to modify existing pseudouridines, and the 3_ ends of the RNA are ligated to linker-adapters. Next, the RNA fragments are reverse-transcribed to cDNA; however, upon encountering CMC-modified pseudouridines, reverse transcription is halted. cDNA strands of 20_80 nt are isolated and processed into cDNA libraries using the Ribo-Seq/ARTseq method before high-throughput sequencing
Similar methods: Pseudo-seq, _-Seq, CeU-Seq
Advantages:
- Identifies pseudouridylation sites in ncRNAs
- Single-base resolution
- Uses a regression analysis to compare reads in a specific location between treated and mock-treated libraries (Zaringhalam et al., 2016)
Disadvantages:
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:
Lovejoy A. F., Riordan D. P. and Brown P. O. Transcriptome-Wide Mapping of Pseudouridines: Pseudouridine Synthases Modify Specific mRNAs in S. cerevisiae. PLoS One. 2014;9:e110799
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History: PSI-Seq
Revision by sbrumpton on 2017-06-21 07:50:22 - Show/Hide
Pseudouridine Site Identification Sequencing
PSI-Seq identifies RNA sequences containing pseudouridine sites using high-throughput sequencing (Lovejoy et al., 2014). PSI-Seq uses N-Cyclohexyl-N_-(2-morpholinoethyl)carbodiimide (CMC) to modify pseudouridines selectively, effectively halting reverse transcription. The cDNA libraries are prepared by the ARTseq method.
Briefly, samples are poly(A)-selected, treated with DNase, and fragmented. CMC is added to modify existing pseudouridines, and the 3_ ends of the RNA are ligated to linker-adapters. Next, the RNA fragments are reverse-transcribed to cDNA; however, upon encountering CMC-modified pseudouridines, reverse transcription is halted. cDNA strands of 20_80 nt are isolated and processed into cDNA libraries using the Ribo-Seq/ARTseq method before high-throughput sequencing
Similar methods: Pseudo-seq, _-Seq, CeU-Seq
Advantages:- Identifies pseudouridylation sites in ncRNAs
- Single-base resolution
- Uses a regression analysis to compare reads in a specific location between treated and mock-treated libraries (Zaringhalam et al., 2016)
Disadvantages:Reagents:Illumina Library prep and Array Kit SelectorReviews:Zaringhalam M. and Papavasiliou F. N. Pseudouridylation meets next-generation sequencing. Methods. 2016;107:63-72References:Lovejoy A. F., Riordan D. P. and Brown P. O. Transcriptome-Wide Mapping of Pseudouridines: Pseudouridine Synthases Modify Specific mRNAs in S. cerevisiae. PLoS One. 2014;9:e110799