bacTRAP profiling of astrocytes in five mouse brain regions in wildtype controls and hippocampus region in a Alzheimer's disease mouse model
We experimentally generated astrocyte gene expression profiles in five major brain regions in mouse: prefrontal cortex (P), hippocampus (H), amygdala (A), caudate nucleus (C) and ventral striatum (V) (Figure 1A, Methods), with more than three biological replicates in each region. To derive the differences between the regional transcriptional profiles, we utilized TRAP-seq, a method that enables detailed profiling of actively translated mRNA, rather than just transcribed mRNA, thus better capturing the functional state of the cell. This approach enables us to generate ex vivo region-specific astrocyte signatures. Overall design: Mouse transgenic line Adlh1l1-TRAP (www.gensat.org; gift from Drs. Heintz and Dougherty) and wild type C57Bl/6J mice (JAX strain #000664) were used for this project. Twelve to fourteen-week-old healthy male and female mice were lightly anesthetized with CO2 and the brains were quickly removed and dissected. Sterile and RNase free instruments and buffers were used. Brains were placed in the 0.5mm matrix and following tissue blocks were cut using razor blades: for the prefrontal cortex (Bregma ~3.56 to 1.94mm), with additional removal of the olfactory bulbs using forceps; Bregma ~1.94 to 0.74 mm from which caudate nucleus (dorsal striatum) and ventral striatum, were cut though using forceps, at an ~45% angle in lateral-to-medial plane, avoiding the midline containing basal forebrain; Bregma 0.74 to -0.46 mm tissue block was used to dissect dorsal striatum using forceps, while amygdala was dissected from the ventro-lateral portion of the block Bregma -0.46 to -2.54 mm. The dorsal hippocampus was removed from each block using the hippocampal tool. A block from Bregma -2.54 to -4.90 mm was used to remove the remaining ventral hippocampus using the hippocampal tool. For each brain region, three technical replicates were processed to gather the astrocyte-specific mRNA for sequencing. Technical replicates contained two pooled biological replicates for prefrontal cortex, caudate nucleus, and hippocampus, and six biological replicate samples for ventral striatum and amygdala. Males and female samples were pooled according to the sex. For Alzheimer's disease mouse model, we utilized two mouse lines, Aldh1l1-TRAP and 5xFAD (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax; The Jackson Laboratory, strain # 008730). Immunoprecipitation was performed overnight with the mixture of two GFP monoclonal antibodies. mRNAs were purified using Qiagen Rneasy Micro Plus kit, following the manufacturer's protocol, and quantity and quality were determined with a Nanodrop Spectrophotometer and Agilent Bioanalyser. For each sample, mRNAs were amplified using the Ovation RNA-seq System V2 (NuGEN). Library preparation and amplification were done by the Rockefeller University Genomic Facility, and Illumina HiSeq platform was used.