Ribosome Footprinting (RiboSeq) analysis of mRNA translation in Dis3l2 knockout mESCs
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PROVIDER: PRJNA562279 | ENA |
REPOSITORIES: ENA
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Project description:Input mRNAs and ribosome protected fragments (RPFs) from Heterozygout (Control) and Dis3l2 knockout (KO) mESCs cultures were analyzed by TruSeq Ribo Profile (Illumina) library preparation and high throughput sequencing
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Project description:We performed ribosome profiling of polysomes (Poly-RIBOseq) to monitor translation of RNAs associated with polysomes in brain extracts from a mouse model Keywords: ribosome profiling, translation, polysome profiling, Poly-RIBOseq
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Project description:We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs. Lysates of hand-dissected Drosophila mature oocytes (containing ~540 M-NM-<g of total RNA) were subjected to separation by velocity sedimentation through sucrose gradients. In this way, free mRNAs (present in RNPs fraction) or those comigrating with ribosomal subunits (40S or 60S+80S fractions) or with varying numbers of bound ribosomes (low polysomes (2-4 ribosomes), medium polysomes (5-9 ribosomes), and heavy polysomes (more than 10 ribosomes) can be separated based on their size and collected as sucrose gradient fractions. To compare quantitatively the levels of every mRNA across the polysome gradient fractions, we added 5ng of S. cerevisiae mRNA as an exogenous spike-in to each of the six fractions of interest: RNPs, 40S, 60S+80S, low polysomes, medium polysomes and heavy polysomes. RNA was extraced from these fractions, follwing proteinase K treatment, by hot acid phenol method. In case of unfractionated lysates, RNA was extracted using TRIzol (Invitrogen) according to manufacturerM-bM-^@M-^Ys instructions. mRNA-seq samples were prepared from 1 M-NM-<g of total RNA (in case of sucrose gradient fractions and unfractionated lysates) and subject to Illumina based sequencing. Puromycin-treated lysates of mature oocytes or 0-2h Drosophila activated eggs (containing ~540 M-NM-<g of total RNA) were also subjected to separation by velocity sedimentation through sucrose gradients. Puromycin causes premature termination of elongating ribosomes and thus it can be used to determine whether the mRNAs co-sedimenting with the polysomal peaks (defined here as M-bM-^IM-%5 ribosomes) were actively engaged in translation. As an independent approach to assess translation and obtain information on the position of ribosomes on mRNAs, we employed ribosome footprinting. In addition to analyzing the same samples, as by polysome profiling, we also analyzed png mutant activated eggs by ribosome footprinting. Ribosome footprint profiling measures the number of ribosome-protected fragments (RPFs) derived from the mRNAs of each gene, resulting in a singular value of translational efficiency (TE) for each gene (TE=RPF/RNA).
2014-06-12 | E-GEOD-52799 | biostudies-arrayexpress
Project description: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 micropeptideM-bM-^@M-^Pencoding genes in vertebrates, providing an entry point to define their function in vivo. Ribosome profiling experiments at five timepoints across zebrafish development in WT embryos
2014-04-04 | E-GEOD-53693 | biostudies-arrayexpress
Project description:Ribosome profiling (RiboSeq) (maps positions of translating ribosomes on the transcriptome) and RNASeq (quantifies the transcriptome) analysis of murine 17 clone 1 (17Cl-1) cells infected with Murine coronavirus strain A59 (MHV-A59). Samples comprise 1 and 8 h mocks, 1, 2.5, 5 and 8 h post infection timecourse, for each of RiboSeq with cycloheximide (CHX), RiboSeq with harringtonine (HAR), and RNASeq, performed in duplicate (6 x 3 x 2 libraries); RiboSeq CHX, RiboSeq HAR and RNASeq at 1 h post infection for high multiplicity of infection (3 libraries); and 1 \long-read\ library for 5 h post infection RiboSeq CHX to test for larger-than-normal ribosome footprints.
2016-02-08 | E-MTAB-4111 | biostudies-arrayexpress
Project description:We applied ribosomal footprinting experiment followed by RNA-seq in order to detect circular RNA associated with ribosomes in fly heads.
2017-03-24 | GSE79626 | GEO
Project description:We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs.
2014-06-12 | GSE52799 | GEO
Project description: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.
2014-04-04 | GSE53693 | GEO