Illumina HiSeq 4000 paired end sequencing; RNA-seq of neuronal SOX2-ablated murine suprachiasmatic nuclei against wild-type littermate controls
RNA-seq of neuronal SOX2-ablated murine suprachiasmatic nuclei against wild-type littermate controls
Tissues were harvested at prescribed circadian time on the third day of constant darkness. Mice were killed by cervical dislocation and their brains were rapidly dissected under dim red light. Brains were sectioned in ice-cold diethyl pyrocarbonate (DEPC)-treated phosphate buffered saline,pH 7.4, with an oscillating tissue slicer to obtain an 800μm thick coronal slice containing the SCN. The 800μm coronal slice containing the SCN was placed on a dry ice-chilled glass slide and the SCN was microdissected using a sharp scalpel blade. Each sample represented a pool of SCN tissues from 3 mice. RNA extraction was performed using the RNeasy Micro Kit according the manufacturer's instructions. RNA samples were submitted to the Génome Québec Innovation Centre at McGill University (Montréal, Canada) for RNA-seq library preparation. Total RNA was quantified using a Nanodrop Spectrophotometer ND-1000 (NanoDrop Technologies) and its integrity was assessed on a 2100 Bioanalyzer (Agilent Technologies). Libraries were generated from 250 ng of total RNA as follows: mRNA enrichment was performed using the NEBNext Poly(A) Magnetic Isolation Module (New England BioLabs). cDNA synthesis was achieved with the NEBNext RNA First Strand Synthesis and NEBNext Ultra Directional RNA Second Strand Synthesis Modules (New England BioLabs). The remaining steps of library preparation were done using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs). Adapters and PCR primers were purchased from New England BioLabs. Libraries were quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies) and the Kapa Illumina GA with Revised Primers-SYBR Fast Universal Kit (Kapa Biosystems). Average size fragment was determined using a LabChip GX (PerkinElmer) instrument.
Experimental Factor: genotype
Sox2fl/fl
Experimental Factor: time
CT6