Re-evaluating Microglia Expression Profiles Using RiboTag and Cell Isolation Strategies

Transcriptome profiling is widely used to infer functional states of specific cell types, as well as their responses to stimuli, to define contributions to physiology and pathophysiology. Focusing on microglia, the brain macrophages, we report here a side-by-side comparison of classical cell sort-based transcriptome sequencing and the ‘RiboTag’ method that avoids cell retrieval from tissue context and yields translatome sequencing information. Conventional whole cell microglia transcriptomes were found to be significantly tainted by artifacts induced by tissue-dissociation, cargo contaminations and transcripts sequestered from ribosomes. Conversely, our data highlight the added value of RiboTag profiling to assess the accuracy of Cre transgenic mice. Collectively, this study indicates method-based biases, reveals observer effects and establishes RiboTag-based translatome profiling as a valuable complement to standard sort-based profiling strategies. overall there are 67 samples, from total of 3 experiments. in each experiment there were at least 3 biological repeats (3 individual mice). sorting and IP were performed from the same animal. Controls include isotype control for IP, PBS injection as control for LPS, and non tamoxifent treated animals as another IP contorl.