GSM5014304: P2_Cortex_Dlxpos_RNA; Mus musculus; RNA-Seq
For embryonic timepoints, male hemizygous Dlx6a-Cre mice (Jax stock #008199) were crossed with female homozygous Ai9 or Ai14 mice (Rosa-LSL-tdtomato, Jax stock # 007909 or 007914) and checked daily for the presence of a vaginal plug, with the morning a plug was found being considered embryonic day 0. Embryos were screened using a fluorescent microscope for the presence of tdTomato. Embryos were harvested from pregnant dams and relevant brain regions were dissected in ice-cold Leibovitz's L-15 medium. Pooled tissue from several embryos were dissociated using the Neural Tissue Dissociation Kit (T) (Miltyeni Biotec, PN-130-093-231) according to the manufacturer's instructions. Dissociated cells were then filtered through a 70 um filter, centrifuged at 300xg for 5 min at 4C, and, where cell-sorting was required, resuspended in 1% BSA in PBS for cell sorting of tdTomato+ cells. Sorting was performed on a Sony SH800S cell sorter with a 100 µm chip.  For postnatal timepoints, male hemizygous Dlx6a-Cre mice were crossed with female homozygous INTACT mice. Nuclei for scRNA-seq were isolated as described previously with some modifications. Briefly, brains were harvested, sectioned coronally on a stainless steel mouse brain slicer (Zivic Instruments), and regions of interest were dissected in ice cold Homogenization Buffer (HB) (0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH, 1mM DTT, 0.15mM spermine, 0.5mM spermidine). Dissected tissue was then transferred to a 2 ml dounce homogenizer containing HB + 0.3% IGEPAL-CA630 + 0.2U/µl RNAse inhibitor and homogenized with 10 strokes of pestle A and 10 strokes of pestle B. Nuclei were then filtered through a 30 μm filter and centrifuged at 500xg for 5 minutes at 4C. The pellet was resuspended in 1% BSA + 0.2U/µl RNAse inhibitor in PBS and centrifuged again at 500xg for 5 minutes at 4C. The pellet was then again resuspended in 1% BSA + 0.2U/µl RNAse inhibitor in PBS an filtered through a 40 μm filter for sorting of GFP+ nuclei on a Sony SH800S cell sorter with a 100 µm chip. Nuclei were collected in 1% BSA + 0.2U/µl RNAse inhibitor for input to 10X Genomics scRNA-seq.  (flox-Sun1-eGFP, Jax stock #021039) to yield Dlx6a-Cre;INTACT offspring. scRNA-seq libraries were prepared using the Chromium Single Cell 3' Solution v3 (10X Genomics). Libraries were sequenced using a Nova-Seq S2 100 cycle kit (Illumina).