title: E2_E2.null_fat_FD_5
organism: Drosophila melanogaster
description: Protocols: Drosophila melanogaster larvae were reared on standard molasses-cornmeal medium at 24⁰C and a 12 hr light/dark cycle with lights on at 0800hr. Larvae were reared to 72 ± 2 hrs old third instar and collected for treatment. The food-deprived treatment group was food-deprived on PBS-soaked filter paper for 4hrs immediately before dissections, the fed treatment group was placed food for 4 hrs. Central nervous systems (brain and brainstem, CNS), fat bodies, and intestines of 76 ± 2 hrs old third instar individual larvae were manually dissected in Schneider's Insect Medium (Sigma-Aldrich S01460) and immediately placed on dry ice. Tissues of four individuals were pooled for intestine and fat body samples and six individuals for CNS samples. Total RNA was extracted using the Arcturus PicoPure RNA Isolation Kit (catalog no. KIT0204; Applied Biosystems) and the RNase-free DNase set (catalog no. 79254; Qiagen), with the following modifications from the manufacturer's protocol: 100 μL of RNA extraction buffer was added to each sample and samples were incubated at 42 °C for 30 min. The manufacturer's protocol for CapSure Macro LCM Caps was then followed starting at step 2, including the optional DNase treatment step. Samples quality control and integrity check was performed on a Bioanalyzer 2100 (Agilent) with the Agilent RNA 6000 nano kit. Libraries were prepared with the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (E7760) following PolyA enrichment for mRNA.