GSM7918347: Cux2-Control; Mustela putorius furo; RNA-Seq
E37 ferret embryos previously electroporated at E34 were obtained by cesarean section under deep anesthesia with sodium pentobarbital, anesthetized, placed in ice-cold phosphate buffered saline (PBS) (1X) and decapitated. The brains were extracted, the meninges removed in cold Leibovitz's (1X) L-15 medium with L-Glutamine, without pH indicator (Gibco), and the parietal cortical region was dissected from the electroporated hemispheres under a fluorescence dissecting scope (Leica MZFLIII) using microsurgical knives (MSP) in ice-cold L-15 medium. Microdissected cortical tissue blocks containing GFP+ cells from control or Cux2-OE animals were transferred to 2 ml DNA LoBind tubes (Eppendorf). Tissue pieces were enzymatically dissociated for 35 min at 37ºC using MACS Neural Tissue Dissociation Kit P (Miltenyi Biotec) according to the manufacturer's instructions. Cells were mechanically suspended using a wide-tipped, fire-polished Pasteur pipette (Fisher Scientific) followed by a P1000 pipette (Gilson). Cell suspensions were filtered to new 2ml DNA LoBind tubes through 40μm PluriStrainer Mini (PluriSelect), pre-wet with Hank's Balanced Salt Solution (1X) without CaCl2/MgCl2/pH indicator (HBSS, Gibco).  Cells were centrifuged (Spectrafuge 24D, Labnet) 2 times for 5min at 1000rpm, and cell pellets were resuspended in 100 μl of PBS pH7.4 (1X) without CaCl2/MgCl2 (Gibco) containing 0.04% Bovine Serum Albumin (BSA, Sigma-Aldrich). Finally, cell suspensions were re-filtered through 40μm PluriStrainer Mini pre-wet filter. Cell death was estimated using trypan blue 0.4% stain (Thermo Fisher Scientific) using a hemocytometer (BLAUBRAND Neubauer, BRAND) on an inverted microscope (Leica DM IL). Debris-free suspensions with cell viability over 90% were immediately FAC-sorted. Before fixation, cells from control or Cux2 overexpressed embryos were pooled together to meet the minimum specification of 105 starting cells of the protocol. In total, approximately 105 GFP+ cells from control embryos, and 2.5 x 105 GFP+ cells from Cux2-OE embryos, were retrieved. scRNA-seq libraries were generated according to Parse Biosciences protocols: Evercode Cell Fixation v2 and Evercode WT v2. For cell fixation, cell suspensions were centrifuged for 10 min at 200g at 4°C in 15 mL BSA-coated Falcon tubes and resuspended into Cell Prefixation Buffer with BSA 7.5% (Gibco) in 15 mL BSA-coated Falcon tubes. Cell viability was inspected as indicated above before proceeding. No significant cell death or damage was observed. Cell suspensions were incubated in fixation solution followed by addition of permeabilization solution and neutralization buffer, according to the manufacturer's instructions. Cell suspensions were then centrifuged for 20 min at 200g at 4°C and cell pellets resuspended in 150 µL of nuclear buffer and filtered through a 40μm filter (FALCON). DMSO was added to the cell suspensions for cryopreservation and their cell concentration was estimated with trypan blue 0.4% stain (Thermo Fisher Scientific) using a hemocytometer (BLAUBRAND Neubauer, BRAND) on an inverted microscope (Leica DM IL). Cell suspensions were frozen using a Mr. Frosty Freezing container (Sigma-Aldrich) at -80°C until further use. Libraries were prepared using Parse protocol and sequenced on Novaseq 6000 instrument, and were prepared from n = 3 control or n = 4 Cux2-OE embryos from a single litter. scRNA-seq libraries were generated according to Parse Biosciences protocols: Evercode Cell Fixation v2 and Evercode WT v2