Illumina HiSeq 4000 sequencing; Circadian and Sleep regulation of synaptic RNA
Circadian and Sleep regulation of synaptic RNA
Synaptoneurosomes from mouse forebrain were prepared as described previously (P. R. Dunkley, P. E. Jarvie, P. J. Robinson, A rapid Percoll gradient procedure for preparation of synaptosomes. Nat Protoc 3, 1718-1728 (2008)). In brief, brain was isolated and rapidly cooled to 4°C, washed in ice-cooled sucrose buffer (320 mM Sucrose, 5 mM HEPES, pH 7.4) followed by homogenization with a Teflon-glass tissue grinder using a motor-driven pestle keeping samples cooled. Homogenate was centrifuged at 1000 g for 10 min. 2 ml of the supernatant were loaded over discontinuous Percoll gradients (3%, 10% and 23% Percoll in sucrose buffer) and centrifuged at 31000 g for 5 minutes. The fraction at the interfaces between 3-10% and 10-23% were collected and further centrifuged at 30000 g to pellet synaptoneurosomes. All centrifugation steps were performed at 4°C. All solutions were supplemented with complete protease inhibitor cocktail (Roche®), 0.05mM DTT, 0.1mM PMSF and RNaseOUT 20 U/10 μl (Invitrogen®). Fraction 'forebrain' = forebrain homogenate before any gradient fractionation, fraction 'synapse' = final synaptoneurosome fraction (pellet obtained from the two pooled Percoll fractions).  200 μl of homogenate were used for total RNA extraction. Briefly, tissue lysate in QIAzol Lysis Reagent (Quiagen ®) was vortexed for few seconds. 0.2 ml of chloroform was added to lysate and mixed by vigorous shaking for 15 sec. The mixture was incubated at room temperature for 10 min and centrifuged at 18,000 g for 20 min at 4°C to separate phases. The upper aqueous phase containing RNA was carefully aspirated to a fresh, nuclease-free tube. The RNA was then precipitated by adding one volume of isopropanol and centrifuged at 18,000 g for 20 min at 4°C. The RNA pellet was washed with 70% ethanol, air-dried and dissolved in nuclease-free water.  Frozen synaptoneurosome pellets (approximately 500 μl) were processed with High Pure RNA Isolation Kit Roche®.  Samples were mixed with 600 μl of Lysis Binding buffer and further steps were carried out according to the manufacturer's instructions. The dissolved total RNA was quantified by NanoDrop (NanoDrop Technologies) and Qubit® (1.0) Fluorometer (Life Technonologies, California, USA). The quality was assessed with Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany).  PolyA RNA sequencing was performed using 500 μg total RNA. Strand-specific cDNA libraries were prepared using Illumina's TruSeq stranded Sample Prep Kit (125 bp single-read mode) following the manufacturer's directions.
Experimental Factor: time
ZT 12