GSM6217292: NPCs, polysome mRNA, Torin1, biol rep1; Mus musculus; RNA-Seq
Mouse cerebral cortices at E13.5 were dissected and enzymatically dissociated using Accutase (Thermo Fisher) with DNase I (Roche), and triturated using a Pasteur pipette to create a single-cell suspension. Cortical cells were cultured in medium containing DMEM/F12 (Gibico, Cat#11330-032), GlutaMax supplement (Gibico, Cat#35050061), Penicillin/Streptomycin (1x Thermo Fisher, Cat#15070063), B-27 supplement (1x, Thermo Fisher, Cat#12587010), bFGF (5ng/ml, Thermo Fisher, Cat#13256029), EGF (5ng/ml, Thermo Fisher, Cat# PHG0311). The cultures maintained in a humidified incubator at 37℃ with constant 5% CO2 supply. The next day, cells were treated with Torin 1 (250nM, Selleck, Cat#S2827) or DMSO (Sigma, Cat#D2650) for 2 hours, and subsequently with cycloheximide (100ug/ml) for 5 minutes. Cells were then washed in ice-cold DPBS plus cycloheximide (100ug/ml) for 2 times, and then lysed in polysome lysis buffer (20mM Tris 7.5,100mM NaCl,5mM MgCl2, 1% Triton X-100, 0.5% sodium deoxycholate, 1mM dithiothreitol, 100ug/ml cycloheximide, SUPERaseIn RNase inhibitor, proteinase inhibitor cocktail). Lysates were kept 10% as input, and the rest layered onto the 10.5 ml sucrose density gradients which prepared in advance and composed of 1.85 ml 50%, 3.65 ml 35%, 3.65 ml 20% and 1.35 ml 20% sucrose density gradients (20mM Tris-HCl ph7.5, 100mM NaCl, 5mM MgCl2, SUPERaseIn, proteinase inhibitor cocktail). To improve the quality of polysome preparations, after pouring each layer, keep the tubes in -80℃ freezer until complete freezing before adding the next sucrose layer, and then let the gradients thaw out in a fridge. Gradients were centrifuged at 36,000 rpm for 2 hours at 4℃ using SW41Ti rotor with “low” brake option. Fractions corresponding to mRNA associated with >3 ribosomes were collected. RNA was extracted with TRIzol reagent. cDNA libraries were prepared for sequencing using standard Illumina protocols