Total and dendritic RNA-sequencing from primary mouse cortical neurons

Activity-dependent protein synthesis is critical for determining changes in dendritic proteomes underlying brain function, yet the mechanisms governing these changes are lacking. Here, we combined proximity-based labeling of dendritic transcriptome, translatome, and proteome to study the dynamics of RNA regulation in activated synapses. We discovered that depolarization leads to a switch from RNAs translated under basal conditions to new translation of previously unrecognized subsets of depolarization-dependent transcripts. Dynamically regulated RNAs bound by specific regulatory proteins, including NOVA1, FMRP, and ELAVLs, rapidly generate proteins with roles in mitochondrial regulation, RNA metabolism, translational control, and synaptic signaling in dendrites. Knockdowns of activity-induced dendritic RNAs altered neuronal physiology, underscoring how dynamic switches in the regulation of RNAs encoding coordinated sets of proteins underlie synaptic plasticity. For the UV-crosslinking experiments, resting and depolarized neurons were treated with cycloheximide (Chx) and biotin and then were washed twice with 1x PBS supplemented with 100 μg/ml CHX and crosslinked on 150 mm plates on ice in 1x PBS with CHX with one pulse of 400 mJ/cm2 and one pulse of 200 mJ/cm2. Cells were then immediately scraped in fresh 1x ice-cold PBS with CHX and centrifuged at 7000 rpm for 5 minutes at 4°C. The pellets were either flash frozen or handled for the downstream steps as described previously by Kaewsapsak et al. (eLife, 2017) and Hendrickson et al. (Genome Biol., 2016). The pellets were lysed in 0.5 ml of ice-cold RIPA buffer (50 mM Tris (pH8), 150 mM KCl, 0.1% SDS, 1% Triton-X, 5 mM EDTA, 0.5% sodium deoxycholate, 0.5 mM dithiothreitol (DTT),  1x EDTA-free protease inhibitor cocktail, and 100 U/ml RNaseOUT for 10 minutes on ice. The lysates were then briefly sonicated on the Branson® digital sonifier using 10% amplitude for 3 seconds, during which they were held in cold metal blocks, and clarified by centrifugation at 15,000 g for 10 minutes at 4°C. Samples were diluted by adding 0.5 ml of Native Lysis Buffer (NLB) (150 mM KCl, 25 mM Tris pH7.5, 5 mM EDTA, 0.5 % NP-40, 0.5 mM DTT, 1x protease inhibitor, 100 U/mL RNaseOUT). 15% of the lysate was kept as input RNA. The remaining lysate was added 50 µl of RIPA:NLB equilibrated C1 magnetic beads. The pulldown was allowed to proceed overnight at 4°C. Beads were then washed briefly at 4°C with ice-cold: 1) RIPA buffer twice, 2) high salt buffer (1 M KCl, 50 mM Tris, pH8, 5 mM EDTA), 3) urea buffer (2 M Urea, 50 mM Tris, pH8, 5 mM EDTA), 4) RIPA Buffer, 5) 1:1 RIPA: NLB, 6) NLB, and 7) TE (10 mM Tris, pH7.5, 1 mM EDTA). The inputs and beads were then treated with sarkosyl and Proteinase K (2% N-lauryl sarkosyl, 10 mM EDTA, 5 mM DTT, in 1x PBS, supplemented with 200 µg of Proteinase K (Roche) and 4 U of RNaseOUT) at 42°C for 1 hour followed by 55°C for 1 h prior to RNA isolation to digest the biotinylated proteins and free the bound RNAs.