Define 5′ and 3′ UTR Landscapes
A genome-wide detection of known and novel transcription start sites and poly-A sites.
Single Nucleotide Resolution
Both transcription start sites and poly-A sites are detected with single nucleotide resolution.
Identify Alternative Promoters (5′ only)
5′ End-Seq detects alternative promoters for all endogenous genes.
High Confidence Poly-A Sites (3′ only)
3′ End-Seq data analysis enriches the data sets for high confidence poly-A sites by not calling sites that map to a genomic A-rich region.
Transcription Start Site (TSS) is the location of the 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 active transcript end detection, enrichment and mapping, and identifies known and novel transcription start sites at single nucleotide resolution.
Poly-A sites (PAS) and the 3′ UTR are involved in mRNA regulation and stability. Both PAS and 3′ UTRs have been implicated in disease and can function as disease biomarkers and drug targets. 3′ End-Seq is a technology for defining 3′ ends of transcripts by sequencing from the PAS. 3′ End-Seq can identify high confidence, known and novel PAS at single nucleotide resolution.
What are the sample requirements for an End-Seq experiment?
Our recommendation is 3ug of total RNA with a RIN score of 7 or higher.
Do you usually recommend biological replicates, and if so, how many?
We recommend running at least duplicates for publication purposes, however triplicates always produce more reliable data.
What if I want to run both 5’ and 3’ End-Seq on my samples?
You will need 3ug of total RNA per end per sample. Protocols can be performed in parallel so the protocol time is still 3 days.
What is included with an End-Seq service?
End-Seq service includes library preparation from total RNA, sequencing and basic data analysis.
What is the turn-around time for End-Seq service?
From the day we receive the sample we estimate 4-6 weeks for delivery of analyzed data.
What is included with an End-Seq kit?
End-Seq kits include most reagents for library preparation, a list of necessary reagents that are not included can be found in the End-Seq protocol.
What are the sequencing parameters?
Libraries generated using the End-Seq method are typically sequenced using standard SE50 or SE75 conditions on the Illumina HiSeq, NovaSeq, or NextSeq platforms. End-Seq libraries are compatible with paired-end sequencing if desired by the user, however due to the small size of typical End-Seq RNA fragments (~200bp), most fragments are fully sequenced in standard single-end formats.
What is the recommended sequencing depth per sample?
Eclipse Bio’s target is 15-20 million reads per End-Seq dataset.
What is the resolution at which sites are detected?
We are able to achieve single nucleotide resolution for both transcription start sites and polyA sites.
Can you detect transcription start sites of non-polyadenylated RNAs?
Yes, we are able to detect transcription start sites of non-polyadenylated RNAs by performing rRNA depletion in place of polyA selection during End-Seq service.
Can End-Seq detect novel sites?
Yes, End-Seq can detect novel and annotated transcription start sites and polyA sites.
Is differential gene expression analysis included in the basic data analysis?
No, for the End-Seq technology this would be considered advanced analysis and charged on an hourly basis.
What is included with End-Seq basic data analysis?
Basic data analysis includes adapter trimming, removal of UMIs, sequence alignments to the genome (with reads aligned to repetitive elements and PCR duplicates removed),
Does End-Seq kit include sequencing?
No, the kit does not include sequencing. If you purchase the data analysis package you would send the raw reads to Eclipse Bio for analysis.