GSM6602195: Ctrl_P15_C1_5; Mus musculus; RNA-Seq
Cortices from P15 Lhx6-Cre;Erbb4+/+;Rpl22HA/HA and Lhx6-Cre;Erbb4F/F;Rpl22HA/HA mice were rapidly dissected in ice-cold RNase-free PBS and immediately homogenized in SynPER Synaptic Protein Extraction Reagent (ThermoScientific) supplemented with 1x cOmplete EDTA-free protease inhibitors (Sigma-Aldrich), 1 mg/ml Heparin (Sigma-Aldrich), 200 U/ml RNAsin (Promega), 100 μg/ml cycloheximide, and 1 mM DTT (Sigma-Aldrich). Synaptosomes were then prepared according to the manufacturer's instructions. The final synaptosome pellets were resuspended in supplemented SynPER, and Igepal-CA630 (Sigma-Aldrich) was added to the samples to a final concentration of 1%. 100 μl of anti-HA magnetic beads (Pierce #88837) were washed in supplemented SynPER and added to the samples for 3-4 h at 4ºC with gentle rotation. After incubation, beads were washed 3 times in ice-cold washing buffer (300 mM KCl, 1% Igepal- CA630, 50 mM Tris-HCl pH 7.5, 12mM MgCl2, 1mM DTT and 100 μg/ml cycloheximide) and eluted in 350 μl of RLT Plus buffer from the RNAeasy Plus Micro kit (Qiagen) supplemented with 2-mercaptoethanol (Bio-Rad). RNA purification of immunoprecipitated RNA was performed using the RNeasy Plus Micro kit (Qiagen) following the manufacturer's protocol. Library preparation for all samples was performed with 1 ng of total RNA using the SMARTer Ultra Low RNAkit for ultra-low RNA. Samples were then sequenced paired-end using an Illumina HiSeq 2500 platform to approximately 60-80 million mapped reads per sample.