Normal goat serum (ngs)

Perchloric acid (PCA), trifluoroacetic acid (TCA), and hydrogen peroxide (H₂O₂) were purchased from Sigma Aldrich (Saint Quentin Fallavier, France). Ammonium bicarbonate and trypsin were obtained from Fluka-Sigma Aldrich (Saint Quentin Fallavier, France) and Promega (Charbonnieres, France), respectively. Water, formic acid (FA), methanol (MeOH) were all ULC-MS grade and purchased from Biosolve (Dieuze, France). Normal Goat serum was obtained from Clinisciences (Nanterre, France). Protein LoBind tube 1.5 mL and Deepwell plate 96/500 μl Protein LoBind were purchased Eppendorf (Le Pecq, France). Oasis HLB μElution Plate 30 μm, was obtained from Waters (Guyancourt, France). Polypropylene vials and Zorbax 300 SB-C18 1 × 150 mm 3.5 μm were both purchased from Agilent Technologies (Santa Clara, CA, USA).

E14.5 heterozygous Mir146a^+/− pregnant mice were injected with BrdU (Thermo Fisher) at 28 mg/kg and dissected 2 h after injection for pulse chase or at P7 for birth dating analysis. Brains were fixed in 4% PFA (Antigenfix, Diapath) during 24 h at 4 °C, washed 3 times in PBS and incubated 24h in PBS–30% sucrose. Brains were then frozen in O.C.T. compound (Tissue-Tek SAKURA) and stored at – 80 °C. Forteen micrometers sagittal sections were cut using a cryostat (Thermo Fisher) and mounted onto Fisher Superfrost Plus slides. Sections were incubated in 2N HCl for DNA denaturation at 37 °C for 30 min then washed 3 times with 0.1% Triton X-100 (Sigma) in PBS. Blocking was realized using 10% Normal Goat Serum (Clinisciences) and 0.1% Triton X-100 in PBS. Slides were incubated overnight at 4 °C with a rat anti-BrdU antibody (ab6326, Abcam). Slides were then washed 3 times with 0.1% Triton X-100 in PBS and incubated with Alexa fluor 488 donkey anti-rat antibody (A21208, Thermo Fisher) at room temperature for 2 h. Slides were finally washed three times in PBS, mounted using the Prolong gold antifade reagent with DAPI (P36935, Thermo Fisher) and scanned in a Nanozoomer 2.0 (Hamamatsu). 250μm × 250 μm images were taken in the neocortex and horizontally binned in 5 layers of equal size; for birth dating 1000 × 800 μm images were used. BrdU⁺ cells were counted in each bin or layer.