Bioinformatic screening
Animal and human samples
Cell culture and transfections
Plasmids, shRNAs, and DNA cloning
Virus preparation
SV exo-endocytosis evaluation and calcium imaging
Pharmacology
Neurite elongation assay
Single-molecule RNA in situ hybridization and immunofluorescence
Quantitative real-time PCR
Library preparation and sequencing
RNA-sequencing analysis
ChIRP DNA analysis
GO and STRING analyses
5′-RACE PCR
ChIRP and RNA interactome analysis
MS identification of protein interactors following ChIRP/RNA interactome analysis
RNA immunoprecipitation
Western blot
Immunocytochemistry
Statistical analysis and graph construction
Pregnant Swiss mice (E14.5) were anaesthetized by intramuscular injections of solution containing 117 μg of ketamine (Anesketin, Eurovet) and 0.7 μg of medetomidine hydrochloride (Domitor, Pfizer) per gram of body weight. Uterine horns were exposed, and a mixture of the required plasmids (1 to 2 μg/μl) was microinjected with the “Fast Green” dye (Sigma-Aldrich) in the lateral ventricles of embryos. Five current pulses (50-ms pulse/950-ms interval) were delivered across the head of the embryos (36 V), targeting the dorsal-medial part of the cortex. After 3 days of expression, the mothers were euthanized, the embryos were dissected, and the brains were quickly perfused with PBS and 4% paraformaldehyde (PFA). After dissection, the brains were post-fixed for 6 to 10 hours in 4% PFA at 4°C. Vibratome sections (100 m) were collected and prepared for imaging. Analysis of cell migration was performed as previously described (23) by dividing the cortical regions in 10 equal bins and by counting the number of positive neuronal nuclei in each bin. Note that also for neurons showed in Fig. 4 (E versus G), there are differences in the migration of neurons in the controls between experiments, as often observed when different control plasmids are used.