characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2017-09-22T17:02:00Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-09-12T16:11:22Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA104293348
characteristics.characteristic: External Id
characteristics.text: European Bioinformatics Institute (EMBL-EBI)
characteristics.characteristic: INSDC center name
characteristics.text: 2017-09-22T17:02:00Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-09-12T16:11:22Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1918326
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-6056:InR_DN_2
characteristics.characteristic: Submitter Id
characteristics.text: 10
characteristics.unit: day
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: fruit fly
characteristics.characteristic: common name
characteristics.text: RU486
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CHEBI_50692
characteristics.characteristic: compound
characteristics.text: Protocols: Fly guts were micro-dissected in ice-cold RNAlater (Qiagen) solution from 10 day old female flies and RNA was extracted using the QIAGEN total RNA isolation kit and quantified on an Agilent 2100 bioanalyser. Sample concentration and purity of RNA was measured on a NanoDrop spectrophotometer, and RNA integrity was assessed on an Agilent 2100 Bioanalyzer. daGS;>InRDN & RNAi protocol: The following stocks were used: UAS-InRDN (K1409A, containing a dominant negative form of insulin receptor leading to reduction of insulin signaling activity) and UAS-GFP-RNAi stocks were obtained from the Bloomington Stock Centre. UAS-fkh-RNAi line was obtained from Vienna stock center (stock number 37062). Transgenes were expressed under the control of the ubiquitous daGS driver (kindly provided by Veronique Monnier). To drive transgene expressions, RU486 (Sigma) dissolved in ethanol was added to a final concentration of 200 μM to fly food containing 10% (w/v) brewer’s yeast, 5% (w/v) sucrose, and 1.5% (w/v) agar. RNA was extracted using the QIAGEN total RNA isolation kit and quantified on an Agilent 2100 bioanalyser. Sample concentration and purity of RNA was measured on a NanoDrop spectrophotometer, and RNA integrity was assessed on an Agilent 2100 Bioanalyzer. Samples were processed using Illumina’s TruSeq Stranded mRNA LT sample preparation kit (p/n RS-122-2101) according to manufacturer’s instructions. Deviations from the protocol were as follows:250ng total RNA was used as starting material. Fragmentation was carried out for 10mins instead of 8mins, and 14 cycles of PCR were used.mRNA was isolated from total RNA using Oligo dT beads to pull down Poly-Adenylated transcripts. The purified mRNA was fragmented using chemical fragmentation (heat and divalent metal cation) and primed with random hexamers. Strand-specific first strand cDNA was generated using Reverse Transcriptase and Actinomycin D. The second cDNA strand was synthesised using dUTP in place of dTTP, to mark the second strand. Full length TruSeq adaptors, containing a T overhang were ligated to the A-Tailed cDNA. Successfully ligated fragments were enriched with 14 cycles of PCR.
characteristics.characteristic: description
characteristics.text: 200
characteristics.unit: micromolar
characteristics.characteristic: dose
characteristics.text: daGS;InRDN>GFP-RNAi
characteristics.characteristic: genotype
characteristics.text: Drosophila melanogaster
characteristics.characteristic: organism
characteristics.text: Drosophila melanogaster
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_7227
characteristics.characteristic: organism
characteristics.text: intestine
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UBERON_0000160
characteristics.characteristic: organism part
characteristics.text: reduced insulin signaling activity
characteristics.characteristic: phenotype
characteristics.text: GFP shRNA
characteristics.characteristic: rna interference
characteristics.text: Drosophila melanogaster
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000383
characteristics.characteristic: sex
characteristics.text: wDahomey
characteristics.characteristic: strain
characteristics.text: InR_DN_2
characteristics.characteristic: title