Vectashield mounting medium

Cells were seeded onto multispot microscope slides (Hendley-Essex) in culture medium in a humidified incubator at 37°C with 5% CO₂. 24 h after seeding, cells were washed twice in PBS and fixed in 4% PFA in PBS for 20 min at room temperature. Cells were washed twice in PBS and permeabilized for 10 min in 10% Triton X-100. Cells were washed five times in PBS and blocked for 1 h in blocking buffer (20% FBS, 1% Tween-20 in PBS). Blocking buffer was aspirated, and cells were incubated in the primary antibody cocktail diluted in blocking buffer for 1 h. Cells were washed twice in PBS, incubated in blocking buffer for 10 min, and washed twice again in PBS before incubation for 1 h in blocking buffer containing fluorescent secondary antibodies (Data S5). Cells were washed twice in PBS, incubated in blocking buffer for 10 min, and washed twice again. PBS was aspirated, and Vectashield mounting medium (Vector Biolabs) was added to cells before application of the coverslip. The coverslip was sealed with nail varnish, and slides were imaged at room temperature using a 63×/1.4 oil-immersion objective on a Leica TCS SP8 confocal microscope controlled with Leica Application Suite X. Images were normalized without altering gamma using Adobe Photoshop CC 2017.

Pregnant female mice were injected intraperitoneally with BrdU (100 mg/kg, Sigma-Aldrich, B5002) at the specified embryonic age between ZT9 and ZT10. Embryos and pups were fixed and sectioned as described above. Tissues were treated with 2N HCl for 25 min at 55 °C, washed 3 times in PBS at room temperature, and incubated in 10% heat inactivated horse serum (Wisent Inc.) in PBST for 1 h. The slides were incubated overnight at 4 °C in fresh blocking solution containing rat anti-BrdU antibody (1:1000, Bio-Rad Laboratories, Hercules, CA, USA, OBT0030G) and rabbit anti-MeCP2 antibody (1:200, EMD Millipore, Burlington, MA, USA, 07-013). The next day, tissues were washed 5 × 5 min with PBST and incubated for 2 h at RT in the dark with the appropriate secondary AlexaFluor antibody (1:1000, ThermoFisher Scientific) diluted in blocking solution. Sections were washed 5 × 5 min with PBST, followed by one PBS rinse. Free-floating sections from P0 and P7 animals were mounted onto glass microscope slides. Slides were coverslipped with Vectashield Mounting Medium (Vector Biolabs) and sealed with nail polish.

Tissues were washed 5 × 5 min in PBS (pH 7.4) with 0.1% Triton X-100 (PBST), incubated for 1 h at room temperature (RT) in blocking solution (10% heat inactivated horse serum (Wisent Inc., Saint-Jean-Baptiste, QC, Canada) in PBST), and incubated overnight at 4 °C in fresh blocking solution containing the primary antibody (Goat anti-GFP, 1:1000, Eusera, Edmonton, AB, Canada, EU3; Rabbit anti-SOX2, 1:300, Abcam, Cambridge, UK, ab97959). The next day, tissues were washed 5 × 5 min with PBST and incubated for 2 h at RT, protected from light, with the appropriate secondary AlexaFluor antibody (1:1000, ThermoFisher Scientific, Waltham, MA, USA) diluted in blocking solution. Sections were washed 5 × 5 min with PBST, incubated for 10 min in DAPI (1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) diluted in PBS, washed twice in PBS, and mounted onto glass microscope slides. Slides were coverslipped with Vectashield Mounting Medium (Vector Biolabs, Malvern, PA, USA) and sealed with nail polish.