Whole heads of Rh1-GAL4/P{UAS-dPABP-FLAG} flies were seperated from bodies using liquid nitrogen freezing and sieve (0.710 mm) seperation. About 200 fly heads were fixed in 1 ml of 1xPBS (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl) containing 1% formaldehyde and 0.5% NP40 for 30 min at 4¡æ. After fixation, 140 ul of 2 M glycine was added and the sample incubated at 4¡æ for 5 min. The heads were rinsed 3 times with 1xPBS and homogenized in 0.8 ml of homogenization buffer. To prepare homogenization buffer (HB), a solution of 150 mM NaCl, 50 mM Hepes buffer at pH 7.6, 1 mM EGTA, 15 mM EDTA, and 10% glycerol was treated with diethyl pyrocarbonate. Immediately before use, vanadyl ribonucleoside complex (Sigma), SUPERase-In (Ambion), and a protease inhibitor cocktail tablet (Roche) were added to the solution to final concentrations of 8 mM, 50 U/ml and 1 tablet/50 ml, respectively. The homogenate was sonicated and cleared by centrifugation at 13,000 xg for 10 min. RNA bound to the FLAG-tagged PABP was recovered by co-immunoprecipitation using anti-FLAG-M2 affinity agarose beads (Sigma). Before use, the agarose beads were washed four times with HB at 4¡æ. For co-immunoprecipitation, we gently mixed 0.8 ml of homogenate with anti-FLAG-M2 affinity agarose beads from 100 ul of bead suspension for 2 hr at 4¡æ. The beads were then washed four times with HB at 4¡æ. The RNA::PABP crosslink was then reversed by incubating the beads in 100 ul of elution buffer (50 mM Tris-HCl, pH 7.0, 10 mM EDTA, 1.3% SDS, 50 U/ml SUPERase-In) at 65¡æ for 30 min. To isolate RNA, 100 ul of eluant was mixed with 400 ul of Trizol (Invitrogen) and then mixed with 100 ul of chloroform. The mixture was incubated at room temperature for 10 min and then centrifuged at 12,000 xg for 10 min at 4¡æ. The aqueous supernatant was transferred to a new tube and mixed with 250 ul of isopropanol, chilled at ¨C80¡æ overnight, and centrifuged at 12,000 xg for 10 min at 4¡æ. The RNA pellet was rinsed once with 0.5 ml of 70% ethanol and then dissolved in 20 ul of RNase free water. Targets were produced using standard Affymetrix procedures from all mRNA sample recovered. 10 ug cRNA was generated and used for hybridization. This sample is photorecetor cells-enriched sample 1 from Rh1-GAL4/P{UAS-dPABP-FLAG} flies. Keywords = Drosophila, mRNA tagging, photoreceptor cell-specific genes
