Global Analysis of the Drosophila NELF complex

To determine the physiological targets of the NELF complex, and provide insight into the mechanism of NELF activity in vivo. Keywords: gene expression, transcription elongation, stress response Drosophila melanogaster S2 cells, obtained from the Drosophila Genomics Resource Center, were untreated, or treated with dsRNA for 72 hours, as described in Armknecht, S. et al. (2005) Methods in Enzymology, 392: pp. 55-73. Total RNA was then extracted using the RNeasy RNA extraction kit, with on column DNAse digestion (Qiagen), according to manufacturer’s protocol. Gene expression analysis was conducted using Drosophila Genome 2.0 Genechip® arrays (Affymetrix, Santa Clara, CA). Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3’ Amplification One-Cycle Target labeling kit according to manufacturer’s protocol. For each array, 10ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000 and data was obtained using the Genechip® Operating Software (Version 1.2.0.037) and imported into the Rosetta Resolver system (Version 6.0).