E-SMDB-3537 - Transcription profiling of Drosophila melanogaster adults and embryos

Status
Released on 11 November 2006, last updated on 12 October 2011
Organism
Drosophila melanogaster
Samples (0)
Arrays (2)
Protocols (4)
Description
See describtion in Gerber AP. et al (2005). Biochemical purification: Five grams of adult flies or 2.5 g of embryos were used in each affinity purification. Flies or embryos were suspended in 15 ml of buffer B (buffer A plus 1.5 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonylfluoride [PMSF], 0.5 5g/ml leupeptin, 0.8 5g/ml pepstatin, 20 U/ml DNase I, 100 U/ml RNasin [Promega], and 0.2 mg/ml heparin) in a mortar filled with liquid nitrogen and ground with a pestle to a fine powder. The powder was transferred to a glass-dounce, thawed and dounced until the pestle reached the bottom. The suspension was centrifuged twice at 40C and 10,000 g for 10 min. The fat layer on top was aspirated off after each centrifugation. Cleared extract (12.5 ml) was incubated with 600 5l of a slurry (50% [v/v]) of IgG-agarose beads (Sigma) for 90 min at 4C. The beads were washed once with buffer B for 15 min at 4C, and three times for 15 min at 4C with buffer C (20 mM TrisHCl [pH 8.0], 150 mM NaCl, 1 mM EDTA [pH 8.0], 10% glycerol, 0.01% NP-40, 1 mM DTT, 10 U/ml RNasin). PumHD was released from beads by incubation with 150 Units of AcTEV protease (Invitrogen) for 2 hr at room temperature (RT). RNA was isolated from extracts and from the TEV eluates with TRIZOL reagent (Invitrogen) followed by RNeasy Mini-Kit (Qiagen) purification according to the manufacturer's instructions. Microarray analysis: A pool of four control cDNAs (2 ng of each flp, gal4, lacZ and -Gal) was added to each RNA sample prior to labeling as controls for the labeling procedure. Total RNA (12 5g) derived from the extract and 300 ng or 30% of the affinity-isolated RNA were labeled with Cy3 and Cy5 fluorescent dyes, respectively, following cDNA synthesis with amino-allyl dUTP in addition to the four natural dNTPs using a 1:1 mixture of oligo(dT) and random nonamer primers. The Cy3- and Cy5-labeled cDNA samples were mixed and hybridized to Drosophila DNA microarrays.
Experiment types
transcription profiling by array, RNA IP, all pairs
Contacts
MIAME
PlatformsProtocolsFactorsProcessedRaw
Files
Investigation descriptionE-SMDB-3537.idf.txt
Sample and data relationshipE-SMDB-3537.sdrf.txt
Raw data (1)E-SMDB-3537.raw.1.zip
Processed data (1)E-SMDB-3537.processed.1.zip
Array designsA-SMDB-920.adf.txt, A-SMDB-921.adf.txt
Links