GSM7097967: Japanese quail forebrain cells; Coturnix japonica; RNA-Seq
Neuronal cultures were prepared from forebrains of nine-day quail embryos. In a laminar hood, shells from post-fertilized eggs were gently removed (by breaking the shell at its upper tip). Embryos were isolated from the yolk and placed in Dulbecco's Modified Eagle Media/Nutrient Mixture F-12 (denoted dissociation medium). Tissues were then enzymatically and mechanically dissociated . Briefly, skin, skull and meninges were removed by scissors and tweezers, and forebrains were isolated and placed in a 15 ml conical tube containing dissociation media enriched with Papain (30 U/ml) and DNAse 1 (57 U/ml), and placed at 37 °C for 30 min. Following incubation, the solution was gently removed without disturbing the forebrains. Digested forebrains were washed three times with PBS and after the last wash, PBS was completely removed and replaced by 2 ml of plating medium consisting of neurobasal medium (Gibco, Cat. 21103049) supplemented with 2% B-27 (Gibco, Cat. 17504044), 1% Pen/Strep and 0.25% Glutamax (Gibco, Cat. 35050061). Then, tissue was manually dissociated by gentle trituration of solution with a fire polished glass pipette (x15), followed by a single trituration with a 1000 ml plastic tip (to ensure complete dissociation). This solution was then applied onto a 40 mm cell strainer (placed on a 50 ml conical tube and pre-washed by 1 ml plating medium). Additional plating medium was added to strainer after passing the entire solution, to elute remaining cells from strainer. The filtered solution, containing the dissociated cells, was transferred to sterile tissue-culture grade plastic plates (Corning, 60 mm, Cat. 430166) pre-coated with PDL, and these were placed for one hour in an incubator (37°C, 5% CO2). Following incubation, medium was removed and replaced by fresh plating medium (pre-warmed in the incubator). Plates were placed back in incubator and half the media was replaced every two days by fresh and pre-warmed plating medium. Cultures produced from nine-day old embryos, grown for a week DIV had their medium replaced with dissection-dissociation medium supplemented with 20 U/ml papain. The plate was incubated for 10 minutes in 37C, 5% CO2. Cells were then pipetted thoroughly and moved to 15 ml canonical tube for gentle centrifugation (5 minutes at 300 rcf). Supernatant was removed and replaced with 1 ml DMEM with 10% FBS. Cells were then filtered through a cell strainer (40 µm nylon filter, LifeGene). Cells were then centrifuged again, and medium replaced as before. Cells were counted using a hemocytometer. Survival assay was performed with trypan blue. One RNA single cell library was prepared according to 10X manufacture protocol (Chromium Next GEM Single Cell 3' Library & Gel Bead Kit v3.1, PN-1000268) using 20,000 input cells (force_10000 was used).