GSM2790911: dlx6neg-rep3; Mus musculus; RNA-Seq
After removal from the mother, embryos were stored on ice in Leibovitz`s L-15 Media and 1% Fetal Bovine Serum. Brains were removed from the embryos and imbedded in 1% UltraPure Low Melting Point Agarose and sectioned in 50-micron sections with a vibratome (Leica VT1200S). The MGE region was dissected from each embryo. Tissue from multiple embryos was pooled together before dissociation. Embryonic brain tissue pooled from multiple embryos was dissociated into a single cell suspension using Papain dissociation system (Worthington Biochemical Corporation) according to the manufacturer’s instructions. Postnatal brain tissue was dissociated with 1mg/mL of pronase (Roche, #10 165 921 001) in ice cold prebubbled aCSF (Artificial Cerebrospinal Fluid) for 25 minutes. Fluorescent cells were sorted on a Sony SY3200 sorter with a 100 micron nozzle. Cells were sorted into 96-well plates and immediately frozen on dry ice. FlashTag: Immediately prior to use 10mM CFSE (Life Technologies, #C34554) CellTrace solution was prepared according to the manufacturer's instructions, and 2-3ul was injected into the lateral ventricle of E13.5 wild type mouse embryos. Embryos were harvested 3, 6, 12 and 24 hours post-injection. Smart-seq2 protocol was used for cDNA synthesis and libraries were prepared with the Nextera XT DNA Library Preparation Kit according to the manufacturers instructions. Cells were immediately lysed and mRNAs were released when single cells were sorted into wells with 5X Maxima reverse transcription buffer, dNTP mixture, RNase inhibitors (SUPERase In RNase Inhibitor, Thermo Fisher Scientific #AM2696) and water. We reverse-transcribed the mRNAs using Superscript II Reverse Transcriptase (Thermo Fisher Scientific #18064071), and amplified cDNAs for each cell in individual wells using the Smart-seq2 protocol, with a custom modification where a 12-base cell barcode was included in the 3’-end RT primer. This allowed us to perform multiplexed pooling before library preparation with the Nextera XT DNA sample prep kit (Illumina), and returned 3’ biased data similar to the Drop-seq protocol. We quantified the cDNA libraries on Agilent BioAnalyzer and sequenced them on HiSeq 2500.