characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2014-10-07T17:00:57Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T17:11:54Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2360969
characteristics.characteristic: External Id
characteristics.text: UCL Cancer Institute, University College London, London, WC1E 6BT, United Kingdom
characteristics.characteristic: INSDC center name
characteristics.text: 2014-10-07T17:00:57Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T17:11:54Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS405995
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2316:ChIP-groGFP-2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: Kc167
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005819
characteristics.characteristic: cell line
characteristics.text: fruit fly
characteristics.characteristic: common name
characteristics.text: Protocols: Kc167 and S2R+ cells (obtained from the Drosophila Genomics Resource Centre [DGRC]) were grown in Schneiders Insect Cell Medium (GIBCO) supplemented with 10% FBS (Sigma) and 1% penicillin/streptomycin (Thermoscientific). Cell lines were kept at 25C. For Groucho oligomerization experiments cells were cotransfected with dsRNA against the 3UTR of Gro as well as plasmids containing either Gro-GFP or GroL38D;L87D-GFP. Transfections were performed using Effectene according to manufacturers instructions (Qiagen). 300-600ng DNA and 1 microgram of dsRNA were cotransfected per 1-3 x 10^6 cells. Successfully transfected cells were processed six days post transfection. Approximately 5-7 x 10^7 cells were fixed at room temperature. For Gro and GFP ChIP experiments cells were double crosslinked by incubation with Disuccininmidyl glutarate (DSG-Fisher Scientific; 2mM) for 20 minutes on a rocker followed by treatment with 1% formaldehyde for ten minutes. The fixation reaction was quenched with 200mM Glycine after which cells were washed three times with 1x Phosphate Buffered Saline (1xPBS). This was followed by a ten-minute lysis on ice (Nuclear Lysis Buffer: 50mM Tris-HCL (pH 8.1), 10mM EDTA, 1% SDS, 1xRoche Complete Inhibitor) and the addition of 1.6 volumes of IP Dilution Buffer (20mM Tris-HCL (pH 8.1), 150mM NaCl, 2mM EDTA, 1% TritonX-100, 0.01% SDS, 1xRoche Complete Inhibitor). Chromatin released from lysates was fragmented to a range of 200 to 1000bp using a Diagenode Bioruptor set for 20 pulses (30 seconds ON, 30 seconds OFF). Fragmented chromatin was further diluted (five-fold) in IP dilution buffer and precleared for 3-5 hours with Protein G PLUS-Agarose beads and 3 micrograms of normal rabbit IgG (Insight Biotechnology and New England Biolabs). Following this precleared chromatin was either snap-frozen and stored at -80C or immediately used for ChIP. For each ChIP experiment an aliquot of chromatin deriving from approximately 1 x 107 cells was used. An aliquot of chromatin was set aside to use as an input control. ChIP samples were treated with the appropriate concentration of an antibody along with Protein G PLUS-Agarose beads and kept on a rocker at 4C overnight. After immunoprecipitation the beads were subjected to a wash with IP Dilution Buffer and two washes each with IP Wash Buffers 1 and 2 (IP Wash Buffer 1: 20mM Tris-HCL (pH 8.1), 50mM NaCl, 2mM EDTA, 1% TritonX-100, 0.1% SDS; IP Wash Buffer 2: 10mM Tris-HCl (pH 8.1), 250mM LiCl, 1mM EDTA, 1% NP-40, 1% deoxycholic acid)). They were finally washed with TE Buffer before bound chromatin-protein complexes were eluted (IP Elution Buffer: 100mM NaHCO3, 1% SDS). Eluted complexes were treated with RNase A (Invitrogen) at 37C for an hour to eliminate RNA, 180mM NaCl at 65C for 4-6 hours to reverse crosslinks, and Proteinase K (Roche) at 45C overnight to remove proteins. The aliquot kept aside as input was subjected to the same treatment. Purified DNA for ChIP and input was obtained by phenol-chloroform extraction and ethanol precipitation. The concentration of ChIP and Input DNA was checked on the Qubit fluorometer (Invitrogen). Approximately 20-50ng of the purified ChIP and Input DNA was used to generate ChIP libraries using either the NEBnext ChIP-Seq Library Prep Master Mix Kit (E6240S) or the NEBNext Ultra DNA Library Prep Kit (E7370S) according to manufacturers instructions. Adaptor-ligated DNA was amplified with 15 cycles of PCR and fragments of 250-350 bp selected by agarose gel extraction. Resulting libraries were assessed for concentration and size using the Qubit fluorometer and the Agilent2100 Bioanalyzer.
characteristics.characteristic: description
characteristics.text: transfection
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0000726
characteristics.characteristic: genetic modification
characteristics.text: gro-GFP and gro RNA
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: Drosophila melanogaster
characteristics.characteristic: scientific_name
characteristics.text: ChIP-groGFP-2
characteristics.characteristic: title