Fluorescence-Activated Nuclei Sorting of OPCs

Fluorescence-activated nuclei sorting (FANS) was used to purify Sun1-GFP⁺ OPC nuclei from brain tissue^{55 (link),91 (link)}. Control or Atrx^Sox10Cre forebrain tissue was homogenized in 500 μL homogenization buffer 20 mM Tricine KOH, 25 mM MgCl₂, 250 mM sucrose, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, 0.1% IGEPAL-630, 1× protease inhibitor cocktail (Millipore Sigma Cat# 11873580001), 1 μL/mL RNase inhibitor (Thermo Fisher Scientific Cat# 10777019). Samples were diluted to 7.5 mL with homogenization buffer and filtered through a 40 µm strainer. Filtered samples were layered on top of 7.5 mL cushion buffer consisting of 0.5 mM MgCl_2, 0.88 M sucrose, 0.5 mM DTT, 1× protease inhibitor cocktail (Millipore Sigma Cat# 11873580001), 1 μL/mL RNase inhibitor (Thermo Fisher Scientific Cat# 10777019) and centrifuged at 2800 g for 20 mins at 4 °C. Nuclei were collected as a pellet, incubated for 10 min in 500 μL 4% FBS, 0.15 mM spermine, 0.5 mM spermidine, 1× protease inhibitor cocktail (Millipore Sigma Cat# 11873580001) and 1 μL/mL RNase inhibitor (Thermo Fisher Scientific Cat# 10777019) in PBS and resuspended by gentle pipetting. Nuclei were sorted using a Sony SH800 Cell Sorter and Sun1GFP⁺ nuclei were collected. Total RNA was immediately isolated from GFP+ nuclei with a single cell RNA purification kit (NorgenBiotek Cat# 51800).