The primary somatosensory cortex was microdissected as aforementioned. Cells were further dissociated by incubating micro-dissected samples in 0.5 mg/mL pronase (Sigma, #P5147) at 37°C for 10 minutes, followed by incubation in 5% bovine serum albumin for 3 minutes and manual trituration in ACSF using pulled glass pipettes. Cells were then centrifuged for 10 minutes at 600 rpm and resuspended before filtration using a 70 mm cell strainer (ClearLine, # 141379C). Cells were then incubated for 10 minutes at 37°C with Hoechst (0.1 mg/mL) and isolated using a Beckman Coulter Moflo Astrios FAC-sorter. Singlet Hoechst+cells were sorted according to their Forward and Side scattering properties (FSC and SSC), and their negativity for Draq7TM (Viability dye, Far red DNA intercalating agent, Beckman Coulter, #B25595). 5000 to 10 000 cells were FACsorted for each experiment. Three microliters of C1 Suspension Reagent (Fluidigm) was added to 10 ml of FAC-sorted cells GFP+ cells, yielding a 300-500 cells / ml suspension. 12 ml of this suspension mix were loaded on a C1 Single-Cell AutoPrep integrated fluidic circuit (IFC) designed for 10 to 17 mm diameter-cells (Fluidigm, #100-5780). RNA was extracted using an RNeasy kit (Qiagen, #74034), and quality control was done using 2001 Bioanalyzer from Agilent. cDNA libraries were obtained using SMARTseq v4 kit (Clontech, # 634888) and sequenced using HiSeq 2500 sequencer. SMARTseq v4 kit (Clontech, # 634888)
GEO Accession
GSM3264939