GSE116233
Optimization of Ribosome Profiling Using Brain Tissue from Fragile X Mice
Dysregulated protein synthesis is a major underlying cause of many neurodevelopmental diseases such as Fragile X Syndrome. A very robust technique is required to capture subtle but biologically significant differences in neurological disorders. Ribosome profiling, which is based on deep sequencing of mRNA fragments protected from ribonuclease digestion by ribosomes, is a powerful tool to study translational control. However, it has been mainly applied to rapidly dividing cells where translation is robust and where large amounts of starting material are readily available. The application of ribosome profiling to low-input brain tissue where translation is modest and where gene expression changes between genotypes are expected to be small has not been carefully evaluated. Using hippocampal tissue from wide type and fragile X mental retardation 1 (Fmr1) knockout mice, we show that variable RNase digestion can lead to significant sample batch effects. We also establish GC content and ribosome footprint length as quality control metrics for ribonuclease digestion. We performed ribonuclease titration experiments for low-input samples to identify optimal conditions for this critical step that is often improperly conducted. Our data reveal that optimal RNase digestion is essential to ensure high quality and reproducibility of ribosome profiling especially for low-input brain tissue. Overall design: Ribosome profiling libraries of wild type mouse hippocampus were generated with different concentrations of ribonucleases and sequenced with Illumina Nextseq500. 5 experimental and 51 QC samples.