RNA immunoprecipitation (RIP)

RNA immunoprecipitation was performed following the protocol from Jayaseelan et al. (2011). Briefly, primary hippocampal neurons were cultured 4 days in vitro before being lysed using PLB buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7, 0.5% Nonidet P-40, 1 mM DTT, 200 U/ml RNase OUT, 1 tablet of EDTA-free Complete Mini Protease Inhibitor). Protein-G magnetic beads (Dynabeads – ThermoFisher Scientific) were washed twice in NET-2 buffer (150 mM Tris-HCl pH 7, 100 mM Tris-HCl pH 8, 750 mM NaCl, 5 mM MgCl2, 0.25% NP-40, 20 mM EDTA pH 8, 1 mM DTT, 200 U/ml RNase OUT) and then conjugated with Rbfox1 (1D10) antibody overnight at 4°C. Beads/antibody slurry was washed six times using NT-2 buffer (150 mM Tris-HCl pH 7, 100 mM Tris-HCl pH 8, 750 mM NaCl, 5 mM MgCl2, 0.25% NP-40) and finally resuspended in 900 µl of NET-2 buffer for each sample. Primary neuron lysates were centrifugated at top speed (benchtop centrifuge) for 10 min at 4°C and 100 µl of cleared top lysate were added to each IP sample and incubated at 4°C overnight in rotation. Part of the initial samples (1:10) were collected as Inputs or Total samples. After the incubation, beads were washed six times with NT-2 buffer and then resuspended in 150 µl of Proteinase-K digestion buffer (NT-2 buffer supplemented with: 1% SDS, 1.2 mg/ml Proteinase-K) and incubated at 55°C for 30 min in a thermomixer.

RNA was then extracted by adding an equal volume (150 µl) of buffer saturated phenol-chloroform pH 4.5 with isoamyl alcohol, vortexed and centrifuged at 20.000 g for 10 min. The aqueous upper part was carefully collected in a new tube before adding 150 µl of chloroform, vortexed and centrifuged again. The upper part was again transferred in a new tube and 50 µl of 5 M ammonium acetate, 15 µl of 7.5 M LiCl, 5 µl of 5 mg/ml glycogen and 1 ml of ice cold 100% ethanol were added before placing the samples at −80°C overnight to allow RNA precipitation. Samples were then centrifuged at 20.000 g/30 min at 4°C, washed once with 80% ethanol and centrifuged again. RNA pellets were finally resuspended in 20 µl of RNase-free water.

RNA was polyadenylated using Poly(A) Tailing Kit (AM1350, ThermoFisher Scientific) following the manufacturer instruction.

cDNA was generated using SuperScript III First-Strand Synthesis System (Cat. No 18080–051, ThermoFisher Scientific).

RT-PCR was performed using BioRad iTaq Universal SYBR-green Supermix (Cat.No. 172–5120) in a MX3000P (Agilent Technologies) apparatus with the following program: 95°C for 3 min; 95°C 10 s, 60°C 20 s for 40 cycles; 95°C 1 min and down to 55°C (gradient of 1°C) for 41 cycles (melting curve step).

RT-PCR products were run in agarose 2% gel.

Primers used:

TrkB common forward: 5’-CGTGGTGGTGATTGCATCTG-3’

TrkB.FL reverse: 5’-CCATTGGAGATGTGGTGGA-3’

TrkB.T1 reverse: 5’-CAGTGGGATCTTATGAAACAAAACAA-3’

TrkB.T1-upstream intron forward: 5’-TTTGAGCATGACTTACGTTTCG-3’

TrkB.T1-upstream intron reverse: 5’-CCCAGCCTTTGTCTTTCCTT-3’

Camta1 forward: 5’-CCGGAGTTACAAGAAGTGTGG-3’

Camta1 reverse: 5’-CTTGGTCCTGCTTTTTGGTC-3’

Sirt1 forward: 5’-GAGCTGGATGATATGACGCTG-3’

Sirt1 reverse: 5’-CAGAGACGGCTGGAACTGTC-3’