GSM2409960: D2_9mnths_WaterControl_246; Mus musculus; RNA-Seq
Prior to cell collection, all surfaces and volumes were cleaned with 70% ethanol and RNaseZap (ThermoFisher Scientific) solution followed by dH20. Mice were euthanized, eyes enucleated and placed immediately into ice-cold HBSS. Retinas were dissected from the eyes (4 or 9 mo of age, no axon degeneration confirmed by PPD staining) in HBSS on ice and placed directly into 100 μl of a custom HBSS (Gibco), dispase (5 U/ml) (Stemcell Technologies), DNase I (2000 U/ml) (Worthington Biochemical) and SUPERase (1 U/μl) (ThermoFisher Scientific) solution. All retinas were from eyes that had no glaucomatous axon degeneration by PPD staining and analysis. Retinas were incubated for 20 mins at 37 °C and shaken at 350 RPM in an Eppendorf Thermomixer R followed by gentle trituration using a 200 μl pipette. Samples were blocked in 2% BSA, SUPERase (1 U/μl) in HBSS, and stained with conjugated antibodies against Cd11b, Cd11c, Cd34, Cd45.2, GFAP, Thy1.2 as well as DAPI. This cocktail allowed other retina cell types to be accurately removed during FACS. FACS was performed on a FACSAria (BD Biosciences). Thy1.2+ (and negative for all other markers) RGCs were sorted into 300μl buffer RLT + 1% β-ME, vortexed and frozen at -80°C until further processing.
GEO Accession
GSM2409960