PEAT
Paired-end Analysis of Transcription Start Sites
PEAT characterizes transcription start sites (TSS) in mRNA using a technique similar to TIF-Seq (Ni et al., 2010). First, poly(A)+ RNAs are enriched from total RNA and the caps are removed with TAP. The 5′ ends of uncapped mRNAs are ligated to chimeric linkers containing MmeI restriction endonuclease sites prior to RT. The RT primers also contain an MmeI site, resulting in single-stranded cDNA flanked by MmeI sites. The fragments are PCR-amplified and circularized into circular single-stranded cDNA, which is amplified further by rolling-circle amplification. MmeI is used to cut circular cDNA at the 2 MmeI sites to create linear, double-stranded cDNA fragments that are 93_95 bp long. The fragments are ligated to paired-end adapters, amplified, and sequenced.
Advantages:
- Maps transcription initiation patterns using paired-end sequencing
- Improved accuracy and alignment yield compared to older, single-end TSS mapping strategies
Disadvantages:
- Does not distinguish between capped and noncapped RNA as in TIF-Seq
- Not designed to sequence 3′-UTRs as in TIF-Seq
- May produce chimeric fragments
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Megraw M., Cumbie J. S., Ivanchenko M. G. and Filichkin S. A. Small Genetic Circuits and MicroRNAs: Big Players in Polymerase II Transcriptional Control in Plants. Plant Cell. 2016;28:286-303
Murakawa Y., Yoshihara M., Kawaji H., et al. Enhanced Identification of Transcriptional Enhancers Provides Mechanistic Insights into Diseases. Trends Genet. 2016;32:76-88
References:
Morton T., Petricka J., Corcoran D. L., et al. Paired-end analysis of transcription start sites in Arabidopsis reveals plant-specific promoter signatures. Plant Cell. 2014;26:2746-2760
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History: PEAT
Revision by sbrumpton on 2017-06-21 07:50:24 - Show/Hide
Paired-end Analysis of Transcription Start Sites
PEAT characterizes transcription start sites (TSS) in mRNA using a technique similar to TIF-Seq (Ni et al., 2010). First, poly(A)+ RNAs are enriched from total RNA and the caps are removed with TAP. The 5' ends of uncapped mRNAs are ligated to chimeric linkers containing MmeI restriction endonuclease sites prior to RT. The RT primers also contain an MmeI site, resulting in single-stranded cDNA flanked by MmeI sites. The fragments are PCR-amplified and circularized into circular single-stranded cDNA, which is amplified further by rolling-circle amplification. MmeI is used to cut circular cDNA at the 2 MmeI sites to create linear, double-stranded cDNA fragments that are 93_95 bp long. The fragments are ligated to paired-end adapters, amplified, and sequenced.
Advantages:- Maps transcription initiation patterns using paired-end sequencing
- Improved accuracy and alignment yield compared to older, single-end TSS mapping strategies
Disadvantages:- Does not distinguish between capped and noncapped RNA as in TIF-Seq
- Not designed to sequence 3'-UTRs as in TIF-Seq
- May produce chimeric fragments
Reagents:Illumina Library prep and Array Kit SelectorReviews:Megraw M., Cumbie J. S., Ivanchenko M. G. and Filichkin S. A. Small Genetic Circuits and MicroRNAs: Big Players in Polymerase II Transcriptional Control in Plants. Plant Cell. 2016;28:286-303Murakawa Y., Yoshihara M., Kawaji H., et al. Enhanced Identification of Transcriptional Enhancers Provides Mechanistic Insights into Diseases. Trends Genet. 2016;32:76-88References:Morton T., Petricka J., Corcoran D. L., et al. Paired-end analysis of transcription start sites in Arabidopsis reveals plant-specific promoter signatures. Plant Cell. 2014;26:2746-2760