Dissection and Analysis of Drosophila Larval Brains

All flies were reared at 25°C and kept on standard medium. The mutant Tet alleles are described in ^{5 (link),10 (link)}; the wild-type allele used in all experiments is w¹¹¹⁸. Stocks utilized to examine genetic interactions with Tet were sli²/CyO, and robo1⁴/CyO (Bloomington Drosophila Stock center), and robo2^×123/CyO^{26 (link)}. The material used for all whole-genome analysis was either hand dissected third instar larval brains, or, because some experiments necessitated a large input, dissected anterior parts of larvae including the 3 anterior abdominal segments that contain the brain besides other tissues such as imaginal discs, salivary glands, mouth parts and epidermis. Because Tet is highly expressed in the brain and the nerve cell in discs, but not in the other tissues, we call this the Larval Brain Fraction, LBF. Brains and larvae from wt and Tet-GFP third instar larvae were dissected in cold-PBS supplemented with protease inhibitor, snap frozen on dry ice, and stored at −80°C.