Polysome Profiling Method for Low-Input Human Postmortem Brain

Polysome profiling is a powerful technique used to analyze the association of mRNA with ribosomes, providing insights into the translational status of a cell. It relies on the separation of ribosome-bound mRNAs through sucrose density gradient centrifugation, where the number of ribosomes on an mRNA correlates with its sedimentation rate. While numerous studies have successfully applied this method to cell line and mouse tissue, application to the human postmortem brain remains scarce due to challenges related to sample quality and low concentration of recoverable material. To overcome these challenges, we: 1. Implemented a protocol specifically optimized for low-concentration human post-mortem brain tissue. 2. Implemented a gradient-maker-free method to manually prepare sucrose gradients with tunable sensitivity for low-input samples. 3. Adapted the human brain tissue protocol for neuronal cell lines and mouse brain with minimal modification.