RNA stability

Primary hippocampal neurons for RNA stability studies were obtained by crossing Nes-Cre animals with R26-Rbfox1^flox/flox animals. Embryos at E18.5 stage were dissected and fast genotyped by using EZ Fast Tissue Tail PCR Genotypying Kit (EZ Bioresearch) in order to group Nes-Cre;R26-Rbfox1^flox/+ hippocampi and R26-Rbfox1^flox/+ hippocampi. RNA stability was assessed in hippocampal neurons at 4 DIV using Click-iT Nascent RNA Capture Kit ({"type":"entrez-nucleotide","attrs":{"text":"C10365","term_id":"1535436","term_text":"C10365"}}C10365, ThermoFisher Scientific) following manufacturer’s instruction. Briefly, 0.2 mM 5-ethynyl uridine (EU) ribonucleotide homolog was added to the neurons media to label new nascent RNA for 5 hr (pulse). After 5 hr, the media was replaced with normal media (without EU) for 9 hr (chase). Neurons were lysed at time 0 hr (right after 5 hr EU pulse) and at time 9 hr (after 9 hr of EU free media) and the RNA isolated using Qiagen RNeasy Mini kit (Cat.no 74104) according to manufacturer’s instruction. Biotin azide was then chemically bound to the EU containing RNA molecules and streptavidin magnetic beads were used to capture the newly synthesized pool of RNA. cDNA was generated by using SuperScript VILO cDNA synthesis kit (11754–050, ThermoFisher Scientific).

Real time 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). Delta Ct values were obtained using β-Actin as reference gene.

Student t-test was applied for statistical significance assessment.

Primers used:

TrkB common forward: 5’-AGCAATCGGGAGCATCTCT-3’

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

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

TrkC common forward: 5’-CCTGACACAGTGGTCATTGG-3’

TrkC.FL reverse: 5’-CTTGTCTTTGGTGGGGCTTA-3’

TrkC.T1 reverse: 5’-GACACATCCCCACTCTGGAC-3’

β-Actin forward: 5’-TACCACAGGCATTGTGATGG-3’

β-Actin reverse: 5’-TCTCAGCTGTGGTGGTGAAG-3’