characteristics.text: 2012-06-27T13:31:32Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T15:32:16Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA1464075
characteristics.characteristic: External Id
characteristics.text: Helmholtz Centre for Infection Research
characteristics.characteristic: INSDC center name
characteristics.text: 2012-06-27T13:31:32Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T15:32:16Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS071646
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-855:S2_dLint1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: S2
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0001233
characteristics.characteristic: cell line
characteristics.text: fruit fly
characteristics.characteristic: common name
characteristics.text: Protocols: D. melanogaster cell lines were maintained under standard conditions. ChIPs were performed as described in the Upstate Biotechnology ChIP assay protocol. 100*10^6 Kc cells were fixed in 1% formaldehyde for 10 min at RT. Fixation was stopped by the addition of 240 mM glycine. Cells were harvested, washed in ice cold PBS, resuspended in 1 ml SDS-Lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, protease inhibitors) and incubated for 10 min on ice. Lysates were sonicated using a Bioruptor (Diagenode) to obtain an average fragment length of 0.5 kb and centrifuged (at 4 degrees C, 15 min, 13000 rpm). Shearing of the DNA was analyzed by agarose gel electrophoresis following reversal of crosslinks. The supernatant (chromatin) was subjected to ChIP analysis. 8 microliter of anti-dLint-1 #1 were used per ChIP. 140 microliter of chromatin were used per ChIP, diluted 10x with IP-buffer (16.7 mM Tris-HCl, pH 8.0, 16.7 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, protease inhibitors) and pre-cleared with 40 microliter of pre-blocked ProtG beads (1mg/ml BSA, 4 h) (GE Healthcare) for 30 min with rotation. Incubation with antibodies was performed overnight, prior to incubation for 2 h with 35 microliter of 1:1 ProtG slurry at 4 degrees C. Precipitates were serially washed for 10 min, three times with low salt wash buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton-X-100), three times with high salt wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton-X-100), once with LiCl wash buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate), once with TE buffer. Immunoprecipitates were eluted twice with 250 microliter elution buffer (1% SDS, 0.1 M NaHCO3) for each 15 min at RT and crosslinks were reversed by addition of 20 microliter 5 M NaCl and heating at 65 degrees C overnight. Following addition of 10 microliter of 0.5 M EDTA, 20 microliter 1 M Tris-HCl, pH 6.5 and 2 microliter of 10 mg/ml Proteinase K samples were incubated for 1 h at 45 degrees C. DNA was purified with peqGOLD Cycle-Pure Kit (Peqlab) Generation of Lint-1 specific antibodies The C-terminus (aa 302-602) of dLint-1 was fused to GST by cloning into pGex4T1 vector. The recombinant protein was expressed in E. coli BL21 according to standard procedures. The GST-Lint-1-C-term fusion protein was purified via affinity chromatography using a GSTrap FF column (GE Healthcare) and subsequent ion exchange chromatography using a HiTrap SP HP column (GE Healthcare) on an Akta purifier system (GE Healthcare) according to the manufacturer's instructions. For immunization two rabbits (serum #1 and #2) were injected with 0.5 mg of purified GST-Lint-1-C-term fusion protein each (Peptide Speciality Laboratories, Heidelberg, Germany). The specificity of antibodies was verified by RNA interference in Kc cells and subsequent Western blot analysis of nuclear extracts.
characteristics.characteristic: description
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: Drosophila melanogaster
characteristics.characteristic: scientific_name
characteristics.text: S2_dLint1
characteristics.characteristic: title