RNA-seq of cortical synaptoneurosomes from MNK1 and MNK2 KO mice.
RNA-seq of cortical synaptoneurosomes from MNK1 and MNK2 KO mice.
Samples for mRNA-seq experiment were collected from four-week-old male mice for each knockout and wild-type genotypes, used as biological replicates in the subsequent analyses. Cortex was rapidly dissected and homogenized in oxygenated Krebs buffer (118.5mM NaCl, 2.5mM CaCl2, 1.18mM KH2PO4, 1.18mM MgSO4, 3.8mM MgCl2, 24.9mM NaHCO3, 212.7mM Glucose in diethyl pyrocarbonate (DEPC) treated H2O) with protease and phosphatase inhibitors in a dunce grinder. The homogenate was passed through 2x 100µm pre-wet nylon filter (Merck Millipore, NY1H02500) using an 18G needle, followed by a second filtration with a 5µm pre-wet filter (Merck Millipore, NY0502500). The lysate was centrifuged for 10min 1000g 4°C. The synaptoneurosome pellet was washed 1x in 500µl Krebs buffer. The resulting pellet was snap-frozen in liquid nitrogen and stored at -70°C until further use. Frozen synaptoneurosomes were resuspended in 600 mL TRIzol using a 22G syringe and were centrifuged for 10min 13 000g 4°C. Total RNA was isolated using the Direct-zol RNA microprep kit (Zymo Research, #R2062) and treated with DNAse, following the manufacturer's instructions.  Double-indexed stranded mRNA-Seq libraries were prepared using the ILMN Stranded mRNA Library Prep Kit (Illumina, #20040534), starting from 250 ng of input material according to the manufacturer's instructions.