GSM3602618: CD_refed_1; Drosophila melanogaster; RNA-Seq
Four biological replicates for each experimental condition were used for sequencing, and each replicate consisted of ten brains. For dissections, flies were briefly anesthetized on ice and brains were rapidly dissected in cold PBS before being transferred and frozen to an Eppendorf tube on dry ice. All dissections and RNA extractions were performed on the same day. Fly brains were manually homogenized in 700uL TRIzol + 4uL RiboLock using a plastic pestle and vortexing. RNA was extracted to the aqueous phase by adding 200uL chloroform with vigorous shaking, followed by centrifugation at 10,000 x g for 10 minutes at 4C. A second extraction was performed by adding equal parts chloroform to the aqueous phase, followed by centrifugation at 10,000 x g for 10 minutes at 4C. RNA was precipitated by adding equal parts 100% isopropanol, followed by a 15 minute incubation at room temperature and finally centrifugation at 10,000 x g for 15 minutes at 4C to pellet RNA. Supernatant was removed and the pellet was washed three times in 75% ethanol followed by centrifugation at 7500 x g for 5 minutes after each wash. The pellet was air-dried, and RNA was eluted in 15uL of RNase-free water. Samples were ribo-depleted, DNase treated, and libraries were prepared using the Nugen Ovation Model Organisms kit (0350) by the University of Michigan sequencing core facility. Library concentrations were determined by qPCR.
GEO Accession
GSM3602618