Illumina HiSeq 2500 sequencing; Transcriptome profiling of tdTomato+ CGNPs from cerebellum of Math1-CreERT2 transgenic mice
Transcriptome profiling of tdTomato+ CGNPs from cerebellum of Math1-CreERT2 transgenic mice
Mice were conventionally housed, fed ad libitum, and routinely genotyped by PCR. Timed matings were performed overnight, with the following morning considered E0.5. Pregnancies were detected by measuring female weight gain at E13.5. A subset of pregnant females received a single dose of Tamoxifen (2 mg/100 μl peanut oil, Sigma) by oral gavaging at E13.5. Pregnant females were killed by asphyxiation (CO2), neonatal mice until the age of P7 were killed by decapitation. For transcriptional analyses, CGNPs were harvested from E13.5, E15.5, P0, P7, P14, and P30 tdTomato+ dissected cerebella from offspring from Tamoxifen-treated females. For primary cell cultures, CGNPs were harvested from E13.5, E15.5 and P7 days old cerebella from offspring of non-treated females. Cerebellar dissection was performed using a stereomicroscope. E15-P30 Cerebella were dissociated with a Papain dissociation kit following the manufacturer's instructions (Worhtington). Following papain treatment, ovomucoid was added to stop the reaction. For P14 and P30 cerebella, an additional Percoll gradient step was performed to remove myelin and debris. Cell suspensions were filtered through a 70 μm cell strainer prior to further processing. Cerebellar single cell suspensions (E15.5-P30) were subjected to fluorescenceactivated cell sorting (FACS) for tdTomato+ cells, using a MoFlo Astrios cell sorter (Beckman Coulter). tdTomato+ cells (CGNPs) were collected in 1.5 ml tubes, centrifuged, and snap frozen. RNA was extracted using the NucleoSpin RNA XS kit (Macherey-Nagel), following the manufacturer's instructions. RNA concentration was measured with a QuBit RNA HS assay kit (Thermo Fisher) and analyzed on a bioanalyzer using the Agilent RNA 6000 Pico Kit (Agilent). 2,5-10 ng of RNA per sample was used as input for library preparation. For library prepping, the Clontecht SMARTer stranded total RNA-seq kit-pico was used according to the manufacturer's instructions for mammalian sample input.