characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-03-01T00:17:08Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-03-01T00:17:08Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112294664
characteristics.characteristic: External Id
characteristics.text: Centre for Cancer Cell and Molecular Biology, Barts Cancer Institute, Queen Mary University of London
characteristics.characteristic: INSDC center name
characteristics.text: 2023-03-01T00:17:08Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-03-01T00:17:08Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14405494
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12481:NclWT_3
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: iKras1
characteristics.characteristic: cell_line
characteristics.text: pancreatic ductal cell
characteristics.characteristic: cell_type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: The iCLIP method was performed as previously described in (Lee et al, 2021), with some modifications. Briefly, triplicates of mock transfected or WT Myc-Ncl, S4A Myc-Ncl, S4D Myc-Ncl transfected iKras PDAC cells were seeded onto 10 cm dishes (2 million per dish) and allowed to grow for 48 hrs in the presence of Dox (1µg/ml), before being irradiated once on ice with 150 mJ/cm2 UV light (254 nm) in PBS, using a Hoefer Scientific UV Crosslinker. Cells were then lysed in lysis buffer (50mM Tris-HCl pH 7.4, 100mM NaCl, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate, supplemented with protease inhibitors), cleared, and diluted to a protein concentration of 1mg/ml. RNA was then digested with 0.2 U/ml of RNase I. Myc-tagged Ncl was then immunoprecipitated with 4g anti-Myc-tag antibody (Cell Signaling), pre-conjugated to protein G Dynabeads (Thermo). The RNAs were labelled at the 3’ end using an adapter (/5Phos/AG ATC GGA AGA GCG GTT CAG AAA AAA AAA AAA/iAzideN/AA AAA AAA AAA A/3Bio/) conjugated to an infrared dye to allow the visualization of the complexes on a gel. iKras cells were grown in DMEM supplemented with 10% FBS, 1% Penicillin/Streptomycin, and doxycycline (1µg/ml) to induce KrasG12D expression. Transient transfection with myc-tagged WT, S4A, and S4D Ncl constructs using Lipofectamine 2000 Barcoded and IR dye conjugated immunoprecipitates were resolved by SDS-PAGE and transferred onto nitrocellulose membrane. The region corresponding to 140-200 kDa protein-RNA crosslinked complexes of myc-Ncl was excised to isolate the associated RNAs. Isolated RNAs were reverse transcribed using primers containing experimental 5' barcodes unique to each sample(WT1: NNNN,TCCAC_0,NNN S4A1: NNNN,CCGGA_0,NNN S4D1: NNNN,AGGCA_0,NNN CTRL1: NNNN,GAATA_0,NNN WT2: NNNN,TTTAA_0,NNN S4A2: NNNN,CATTT_0,NNN S4D2: NNNN,ACCTT_0,NNN CTRL2: NNNN,GTATT_0,NNN WT3: NNNN,TGCGT_0,NNN S4A3: NNNN,CAAGT_0,NNN S4D3: NNNN,GCTCT_0,NNN CTRL3: NNNN,AGACT_0,NNN) (3'Adapter: AGATCGGAAG_1,AGCGGTTCAG_2).The cDNAs were then PCR amplified using Solexa P3 and P5 primers, gel extracted, and equal amounts of each amplified library was then combined together into a single mixed pool.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: pancreatic carcinoma
characteristics.characteristic: disease
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: pancreas
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: NclWT_3
characteristics.characteristic: title