iCLIP Analysis of WT, phospho-defective, and phospho-mimicking mutants of mouse Nucleolin in iKras PDAC cells
iCLIP Analysis of WT, phospho-defective, and phospho-mimicking mutants of mouse Nucleolin in iKras PDAC cells
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.