μM DL-AP5. Samples were resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles, and then diluted in 30 mL in Staining Medium (440 mL Leibovitz L-15 medium, 50 mL water, 5 mL 1M HEPES pH 7.3–7.4, 5 mL 100× Pen-Strep, 20 mL 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1 g bovine serum albumin, 100 nM TTX, 20 μM DNQX, and 50 μM DL-AP5), filtered, pelleted (220 × g, 10 min, 4°C), resuspended in 5 mL staining medium, and counted on a hemocytometer (typically ~20–40 M live cells isolated per cortical piece at ~50% viability). Cells were fixed (4% PFA in PBS, 15 min on ice), rinsed twice in staining buffer (SB, PBS, 0.2% w/v molecular biology grade BSA (Gemini Bio-Products), 0.25% v/v RNasin Plus (Promega)), and kept frozen in this buffer at −80°C at 2 million cells/1.5 mL until use. For sor ting specific populations and retrieving RNA from fixed fetal cortical cells, we followed a previously published method for staining and sorting these cells, followed by reversing cross-linking and RNA purification prior to SmartSeq2 RNAseq (Thomsen et al. 2016).
