Cortical neurons were prepared as previously described15 (link). Briefly, E15.5 mouse embryo cortices were dissected and then dissociated in 1× Hank’s Balanced Salt Solution (HBSS) in the presence of 0.1% trypsin (Invitrogen). trypsin treatment was terminated with trypsin inhibitor and triturated in presence of DNase I (Sigma). Neurons were pooled after genotyping and seeded on poly-D-lysine coated dishes. Neurons were maintained in Neurobasal medium containing B27 supplement, antibiotics and glutamine. Neurons were cultured in vitro for 10 days. One third of the medium was replaced with fresh warm medium every two days.
For KCl depolarization, neurons were quieted overnight in 1 μM tetrodotoxin (TTX, Tocris), and then were incubated for 0, 1, 3, 4 or 6 hours in 55 mM KCl as described previously15 (link). ChIP-seq experiments were performed after 0 or 1 h KCl-mediated depolarization. RNA-seq experiments were performed after 0 or 6 hours KCl-mediated depolarization. GRO-seq experiments were performed after 0, 1 or 3 hours KCl-mediated depolarization. 3X depolarization buffer (170mM KCl, 1mM MgCl2, 2 mM CaCl2, 10mM HEPES pH=7.9) was added as 1:3 ratio into culture medium (final 55mM KCl) to induce depolarization of cortical neurons.
For KCl depolarization, neurons were quieted overnight in 1 μM tetrodotoxin (TTX, Tocris), and then were incubated for 0, 1, 3, 4 or 6 hours in 55 mM KCl as described previously15 (link). ChIP-seq experiments were performed after 0 or 1 h KCl-mediated depolarization. RNA-seq experiments were performed after 0 or 6 hours KCl-mediated depolarization. GRO-seq experiments were performed after 0, 1 or 3 hours KCl-mediated depolarization. 3X depolarization buffer (170mM KCl, 1mM MgCl2, 2 mM CaCl2, 10mM HEPES pH=7.9) was added as 1:3 ratio into culture medium (final 55mM KCl) to induce depolarization of cortical neurons.
