GSM7070337
Mice were perfused for 10 minutes with phosphate buffered saline solution before dissection of the cerebral cortex (astrocytes) or whole brain (microglia).  For astrocytes, cortical tissue was roughly chopped then placed in a papain solution (12 units/mL) for enzymatic digestion at 34.5C for 45 minutes. Digested tissue was washed with ovomucoid inhibitor and mechanically digested via trituration.  Cells were spun down and resuspended in PBS with 0.02% bovine serum albumin, then passed through a mesh filter to generate a single cell suspension. Tissue for microglia was dounced, spun down through a Percoll solution, and then resuspended to get a single cell suspension. The final cell suspensions were incubated on various petri dishes (10-20 min/dish) that were coated with antibodies targeting a desired cell population.  Cells bound to each dish were rinsed with PBS, and RNA was harvested immediately with 700 uL of TRIzol before flash freezing in liquid nitrogen. RNA was purified for sequencing using the miRNeasy kit (Qiagen). RNA libraries were constructed with the Ovation V2 system (Tecan) and the NEBNext Ultra RNA Library Prep Kit (New England Biolabs)