GSM5822766: WT1; Mus musculus; RNA-Seq
Brains were dissected and immediately immersed into ice-cold Dissection Buffer (1x HBSS, 2.5 mM HEPES pH7.4, 4 mM NaHCO3 and 35 mM Glucose). Tissues from 4-5 mice were pooled and transferred into Teflon-glass homogenizer with Homogenization Buffer [10 mM HEPES pH7.4, 150 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 0.5 U/mL SUPERase•In (ThermoFisher, AM2696), 0.5 U/mL RNase Inhibitor (ThermoFisher, AM2682) and 2x EDTA-free Halt protease inhibitor cocktail (ThermoFisher, 87786)]. Tissues were then dounced 12 strokes or until no visible clumps of tissue could be seen. Lysate was then transferred into a new RNase-free microcentrifuge tube and cleared by centrifuging at 2000g at 4 oC for 10 minutes. Clear supernatant was then supplemented with 0.6% NP-40 and 18 mM DHPC and incubated on ice for 2 minutes. Samples were centrifuged again at 16,000g for at 4C for 10 minutes and clear supernatant was collected into new microcentrifuge tubes. RNA was extracted with a RNeasy Micro Kit (Qiagen, 74004) RNA quality was determined by a Bioanalyzer 2000 (Agilent) and samples with RIN>9 were used to prepare sequencing libraries. Library were constructed with the SMARTer Ultralow Input RNA for Illumina Sequencing Kit and sequenced on an Illumina HiSeq2500 platform, following the manufacturers' instructions.