Frozen heads were homogenized in a Kontes glass homogenizer (D9938-1SET; Sigma, St. Louis, MO) with 10 ml of Dounce buffer (10 mM β-glycerophosphate, 2 mM MgCl2, 0.5% IGEPAL buffer) and homogenized for ∼2 min with the loose A pestle. Homogenate was passed through a 190-µm nylon net filter (CMN-0185-C; Small Parts). The filter was subsequently rinsed with 2 ml of the same buffer before discarding. Homogenate was subsequently further homogenized gently ∼6–7 times with the tight B pestle, passed through a 20-µm filter (F020N-12-C; Small Parts), brought up to 50 ml by adding sucrose buffer (10 mM β-glycerophosphate, 2 mM MgCl2, 25 mM KCl, 250 mM sucrose). Next, 300 µl of Dynabead A magnetic beads (10001D; Thermo Fisher) were preincubated with rabbit α-GFP antibody (G10362; Thermo), and incubated with the homogenate for 30 min at 4° with gentle agitation. Beads were then captured with a magnet for 15 min and washed five times with 600 µl of sucrose buffer for 5 min at 4° with gentle agitation. Finally, RNA was extracted from bead-bound nuclei using TRIzol (Ambion, Life Technologies), resuspended in RNA-ase free water and DNA-ase treated according to manufacturer’s instructions (Ambion DNA-Free Kit).
