Illumina HiSeq 2000 sequencing; GSM1299080: 5h RPFs column replicate 1 run 1; Danio rerio; RNA-Seq

5h RPFs column replicate 1 run 1

Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide-encoding genes in vertebrates, providing an entry point to define their function in vivo. Overall design: Ribosome profiling experiments at five timepoints across zebrafish development in WT embryos

Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation

Submitted by Gene Expression Omnibus on 22-APR-2014

For ribosome profiling, 50 wild type embryos for each condition were collected at a given stage. Embryos were lysed using 800ul of a mammalian cell lysis buffer containing 100ug/ml Cycloheximide as per the manufacturer’s instruction (ARTseq Ribosome Profiling Kit, RPHMR12126, Epicentre). For nuclease treatment, 3ul of ARTseq Nuclease was used. Ribosome protected fragments were run and 28-29nt fragments were gel purified as previously described in (Bazzini et al., 2012) and cloned according to the manufacturers protocol (ARTseq Ribosome Profiling Kit, RPHMR12126, Epicentre). For RNA input, total RNA was isolated from 400 ul of the 800 ul of clarified extract before ART- seq Nuclease treatment. Poly-A selection was done according to manufacturer guide- lines (Dynabeads mRNA Purification Kit, Cat no.610.06), and RNA fragmented using the Artseq Ribosome Profiling Kit Mammalian protocol. Both RPF and RNA input fragments were cloned according to the Artseq Ribosome Profiling Kit, Mammalian. The final PCR was carried out with an initial 15 second denaturation at 98 °C, followed by 9-12 cycles of 15 seconds at 98 °C, 5 seconds at 55 °C and extension at 72 °C for 10 s. Reactions were separated on a non-denaturing 8% polyacrylamide TBE gel and DNA fragments of the correct size were extracted and sequenced.

Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation

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