Novel astrocytic translatome isolation pipeline uncovers regional and sex-specific differences in mouse brain cortex

Astrocytes, with their intricate morphology, pose challenges for cell-specific RNA isolation using traditional methods, which often result in loss of RNA localised in distal processes. We tackle this issue by adopting the RiboTag method (Sanz et al 2009) in RPL22tm1.1Psam mice. This technique isolates ribosome-associated mRNA (translatome) cell-specifically from crude tissue lysates through ribosomal HA tagging via cell-type specific Cre expression. Despite the widespread use of RiboTag, its detailed adaptation for complex cells like astrocytes, especially in high-throughput scenarios, remains underexplored. Our study fine-tuned this approach for isolating the entire astrocytic translatome from single cortical areas with minimal tissue mass and employed it for RNA-seq analysis of visual and entorhinal cortex translatomes in both sexes. We evaluated two strategies for astrocyte-specific ribosome tagging using RiboTag mice: an AAV.PHP.eB vector with a truncated GFAP promoter, which led to nonspecific neuronal recombination, and an Aldh1l1-CreERT2 driver line, which achieved highly specific astrocytic recombination, both confirmed by immunofluorescence staining. By testing various RiboTag protocol conditions, we optimized the method for maximum high-quality RNA yield, assessed through RT-qPCR and microcapillary RNA electrophoresis. Our optimized method utilizes Aldh1l1-CreERT2 induced recombination, Dounce homogenizer tissue lysis, shortened immunoprecipitation times, and a novel RNA elution technique. We applied this advanced approach to isolate and sequence RNA from astrocytes in the visual and entorhinal cortices of both male and female mice, uncovering significant translatomic variations between the cortical types and minor sex-based differences.