Slabs from the frozen brains were serially cryosectioned at 14 µm onto PEN slides for LMD (Leica Microsystems, Inc., Bannockburn, IL) and a 1:10 Nissl series was generated for neuroanatomical reference. After drying for 30 min at room temperature, PEN slides were frozen at −80°C. Slides were later rapidly fixed in ice cold 70% ethanol, lightly stained with cresyl violet to allow cytoarchitectural visualization, dehydrated, and frozen at −80°C. LMD was performed on a Leica LMD6000 (Leica Microsystems, Inc.) using the cresyl violet stain to identify target brain regions. Samples captured include cortical and subcortical regions and are listed for each brain in the ontological sample map (Suppl. Table 2 ).
Microdissected tissue was collected directly into RLT buffer from the RNeasy Micro kit (QIAGEN Inc., Valencia, CA) supplemented with β-mercaptoethanol. Samples were volume adjusted with RLT Buffer to 75µl, vortexed, centrifuged, and frozen at −80°C. RNA was isolated for each brain region following the manufacturer’s directions. RNA samples were eluted in 14µl, and 1µl was run on the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA) using the Pico 6000 assay kit. Samples were quantitated using the Bioanalyzer concentration output. The average RNA Integrity Number (RIN) of all 1,202 passed experimental samples was 6.3.
Microdissected tissue was collected directly into RLT buffer from the RNeasy Micro kit (QIAGEN Inc., Valencia, CA) supplemented with β-mercaptoethanol. Samples were volume adjusted with RLT Buffer to 75µl, vortexed, centrifuged, and frozen at −80°C. RNA was isolated for each brain region following the manufacturer’s directions. RNA samples were eluted in 14µl, and 1µl was run on the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA) using the Pico 6000 assay kit. Samples were quantitated using the Bioanalyzer concentration output. The average RNA Integrity Number (RIN) of all 1,202 passed experimental samples was 6.3.