GSE60930   Details

GSE Accession GSE60930
Title Genome-wide translational analysis of RAW264 macrophages by ribosome profiling
Submission Date 8/29/14
Last Update Date 11/2/15
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Experiment Type Other; Expression profiling by high throughput sequencing
Contributor Hiroaki,,Sako
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Organism Mus musculus
Organism ID 10090
Organism Synonym house mouse; mouse
Summary Purpose: The aim is to analyze the translational kinetics of the basal and LPS-stimulated conditions of RAW264 macrophages focusing on ribosome density; Method: RAW264 macrophages were cultured at 3.0 x 10^5 cells/ml in media (DMEM, 2mM Glutamine, 10% FBS, 100 units penicillin and 100µg streptomycin/mL) 24 hours prior to harvest. It was confirmed that the confluency of cells never surpassed 80 ~ 90%. Cell were harvested in their basal state or LPS stimulated condition (100 ng/ml for 30 min) and ribosome profiling (Ribo-Seq) and high-throughput mRNA sequencing (mRNA-Seq) were conducted. Ribosome protected fragments (RPF) were sequenced and the density was normalized by mRNA abundance.; Result and conclusion: We discovered where in the ribosome translational arrest ocurs and how this happens. Translational stall was striking at A-site and P-site of ribosomal complex. The other arrest site was observed 3 to 5 residues away from the peptidyl transfer center of the exit tunnel, in which ribosomal density and hydrophobicity showed a significant negative correlation.
Overall Design mRNA and RPF of RAW264 macrophages were deep-sequenced with two independent biological replicates by Ion PGM sequencer
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