title: GSM4834669: UTHT7_ROW11_1; Mus musculus; RNA-Seq
library_construction_protocol: Single-cell RNA sequencing    Embryonic cortex: Tissue collection was performed as in Telley et al., Science 2019. Briefly, pregnant females were sacrificed, and embryos were extracted in ice-cold HBSS either at E15.5 and E17.5 after E14.5 in utero electroporation. Three hundred micrometer- acute coronal brain sections were performed after embedding in 4% low melt- agar using a vibratome (Leica, #VT1000S) under RNase-free conditions. The putative primary somatosensory cortex (S1) was microdissected using a Leica Dissecting Microscope (Leica, #M165FC) and incubated in 0.05% trypsin at 37°C for 5 minutes. Following tissue digestion, cells were incubated in fetal bovine serum and cells were manually dissociated via gentle pipetting. Cells were then centrifuged for 5 minutes at 500 rpm, resuspended in 1 mL FACS buffer (2 mg/mL glucose, 0.1% bovine serum albumin, 1/50 citrate phosphate dextrose, 10 units/mL DNaseI, 1 uM MgCl2, in L15 medium) and filtered using a 70 mm- cell strainer (ClearLine, #141379C), before 10 minute- incubation with Hoechst (0.1 mg/mL) at 37°C .    Postnatal cortex: Tissue collection was performed as in Klingler et al., Curr Biol 2019. Briefly, 600 um acute coronal brain sections were cut on a vibratome and S1 was microdissected as described above in ice-cold oxygenated artificial cerebrospinal fluid (ACSF) under RNase-free conditions. Cells were next dissociated by incubating microdissected samples in 0.5 mg/mL pronase (Sigma, #P5147) at 37°C for 10 minutes, followed by a 3 minute- inactivation in 5% bovine serum albumin, washes in ACSF and manual trituration using pulled glass pipettes of decreasing diameters. Cells were then centrifuged for 10 minutes at 500 rpm, resuspended and filtered using a 70 um- cell strainer (ClearLine, #141379C), incubated for 10 minutes with Hoechst (0.1 mg/mL) at 37°C.    Singlet GFP+/Hoechst+ embryonic and postnatal cells were sorted using a Beckman Coulter Moflo Astrios FAC-sorter according to their Forward and Slide scattering properties, and their negativity for Draq7TM (Viability dye, far red DNA intercalating agent, Beckman Coulter, #B25595). Three microliters of C1 Suspension Reagent (Fluidigm) was added to 10 ul of FACsorted cells, which were captured into 800 well- AutoPrep integrated fluidic circuit (IFC) designed for 10 to 17 um diameter-cells (Fluidigm HT800, #100-57-80), and imaged using the ImageXpress Micro Widefield High Content Screening System (Molecular Devices®).        Bulk RNA sequencing    Brains were collected from E14.5 in utero electroporated miR-122 pups at P7. The primary somatosensory cortex was microdissected following the same procedure as described above with further separation of superficial (SL) and deep (DL) layers. SL and DL cells were dissociated and FACsorted as for single-cell RNA sequencing. Single-cell RNA sequencing  cDNA synthesis and preamplification was processed following the manufacturer's instructions (C1 system, Fluidigm). cDNA libraries were prepared using Nextera XT DNA library prep kit (Illumina), quality control was done using 2001 Bioanalyzer from Agilent, and sequenced using HiSeq 2500 sequencer for E14.5 electroporated pups, and HiSeq 5000 sequencer for E14 and E15.5 electroporated pups with an expected sequencing depth of 1M reads per single cell, and a final mapping read length of 70bp.    Bulk RNA sequencing  RNA from each sample was extracted 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.