GSM2418045: KIF1Bb_SCG_WT3; Mus musculus; RNA-Seq
Total RNA was isolated from snap-frozen SCGs using a modified Trizol extraction protocol as follows. SCG pairs from each animal were thawed and then suspended in 300 ul Trizol (Invitrogen) containing 20 ug/ml Proteinase K (Invitrogen) and incubated at 55°C with shaking for 10 mins. Samples were then cooled on ice, 2 ul of Pelletpaint (Novagen) added, followed by 60 ul chloroform. Samples were then incubated on ice for 5mins, and centrifuged at 20,000g for 10 minutes to separate phases. The upper (aquaeus) phase was then collected and combined 1:1 with isopropanol, mixed vigorously, incubated for 5 minutes on ice and centrifuged for 30 minutes at 20,000g to precipitate RNA. After aspiration of supernatant, the RNA pellets were then washed with 300 ul 75% ethanol and centrifuged for a further 10 mins. After aspiration of the supernatant, pellets were resuspended in 100 ul of water, 10 μl 3M Sodium acetate was added, followed by 250 μl icecold 100% EtOH. Following incubation for 1 hour at -20°C, and centrifugation for 30 mins, suparnatant was aspirated and 300ul of 75% ethanol added to wash the RNA pellets that were then centrifuged for a further 10 mins. Finally, supernatant was completely aspirated from the pellets that were then briefly left to dry at room temperature in a fumehood, and then resuspended in 20 ul water. RNA was then quantified using a Nanodrop, and quality controlled using RNA PICO chips on an Agilent Bioanalyzer. Only samples with an RNA integrity index greater than 7.5 were used for RNA sequencing Truseq v2
GEO Accession
GSM2418045