5´ UTR Sequencing Kit for detecting 5´UTR Variants by constructing mRNA 5´-end enriched RNA-Seq NGS libraries
Reveal 5′ UTR Landscape and Transcription Start Site Usage genome-wide by 5´UTR-Sequencing
5′ End-Sequencing facilitates active transcript 5´ end detection, enrichment and mapping, and identifies known and novel transcription start sites (TSS) at single nucleotide resolution by next generation sequencing.
• Define 5′ UTR Landscape: Genome-wide detection of known and novel transcription start sites
• Single Nucleotide End Resolution: Transcription start sites are detected with single nucleotide resolution
• Identify Alternative Promoters: 5′ End-Sequencing detects alternative promoters for all endogenous genes
• High Throughput and Robust Workflow
• High Reproducibility with accurate data
• Unbiased with high specificity
The transcription start site (TSS) is the location of transcription initiation at the 5′ end of a gene sequence. Knowledge of the exact position of a TSS of an RNA molecule is crucial for the identification of regulatory regions immediately flanking it.
5´End-Seq facilitates precise UTR identification for RNA therapeutics targeting and can be used as a tool for identifying UTR biomarkers associated with disease. 5′ UTR Sequencing can quantify the relative usage of TSS across samples and indicates transcript isoform presence in annotated transcriptomes. Defining the 5′ UTR with 5´End-Seq can also help predict binding factor motifs more reliably.
5′ End-Seq Workflow
Total RNA is enriched for poly(A)+ mRNAs. Poly(A)+ mRNAs are heat fragmented and subsequently 5’ phosphorylated using PNK enzyme, which only uses uncapped molecules as substrate. The phosphorylated mRNA fragments are then digested by exonuclease, leaving behind only mRNA fragments with 5’-caps present. Those are used to prepare an RNA-seq library covering highly enriched sense-strand mRNA 5´ end fragments.
Sample Input: >3 ug Total RNA (>0.1 ug/ul), RIN >7
Sequencing Recommendations: 10-15M reads (SE100) on Illumina
– mRNA purification reagents
– all enzymes and buffers for enrichment of 5´end mRNA fragments
– all enzymes, buffers, adapters and primers for stranded mRNA-seq library amplification and preparation
The 5´UTR Sequencing kit enables the user to prepare 8 high quality mRNA 5´end enriched RNA-seq libraries, ready for sequencing on Illumina.
The Eclipse BioInnovations 5´End-Seq Data Analysis Package uses Fastq files of the raw sequencing reads obtained by you. An HTML report will be generated detailing the following data: Single Nucleotide 5´End Read Coverage Bedgraph, Single Nucleotide 5´End Calls BED, Filtered Single Nucleotide 5´End Calls (false positive 5´ends are filtered out bioinformatically).