ARTSeq Ribosome Profiling Kit (Mammalian)

ARTseq™ Ribosome Profiling Kits

 

Applications

The ARTseq™ Kit produces RNA-Seq libraries from ribosome-protected mRNA to:

• Investigate translational control.
• Measure gene expression.
• Identify translation start sites.
• Predict protein abundance.
• Investigate translational and co-translational processes in vivo.

Ribosome profiling is a novel technique for investigating translational control. The method, based on deep sequencing of ribosome protected mRNA fragments, provides a "snapshot" of the ribosomes active in a cell at a specific time point. This information can in turn be used to determine what proteins are being actively translated in a cell.

Benefits

• Incorporates a rapid and simplified size-exclusion spin-column method to isolate monsomes.
• Compatible with yeast and mammalian samples.
• ARTseq libraries are compatible with Illumina® Cluster Kits for single read, paired-end and multiplex sequencing using the Illumina GAII, HiSeq and MiSeq sequencers.
• Index-compatible: 12 Index Primers included in the kit.

Figure 1 (click to enlarge). Overview of the ARTseq Ribosome Profiling process for converting ribosome protected fragments (RPF) into an RNA-Seq library. Mammalian or yeast cells are treated with cycloheximide (provided by the user) to maintain the in vivo ribosome positions on the mRNAs. Ribosome footprints are generated by digesting cellular lysate with ribonuclease and recovering polysomes or monosomes by ultracentrifugation or size-exclusion chromatography. Following purification, the nuclease protected mRNA fragments are converted into a DNA library for sequencing on any Illumina® sequencing platform.

A

B

Figure 2. Distribution of sequencing reads from ribosome protected RNA or poly(A)+ mRNA samples along a representative yeast transcript (CWP2). A) ARTseq RNA-Seq libraries prepared after Size-exclusion chromatography (SEC) or sucrose gradient centrifugation (Gradient) result in essentially identical distribution of read densities and are enriched for Open Reading Frame (ORF) and devoid of Untranslated sequences (UTR). B) Graphic representation of percent of reads that align to coding, UTR, intron and intergenic regions of the the transcript.