Illumina Genome Analyzer IIx sequencing; Spliceosome iCLIP
Spliceosome iCLIP
Cal51 breast adenocarcinoma cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher) with 10% fetal calf serum (FCS, Thermo Fisher) and 1× penicillin-streptomycin (P/S, Thermo Fisher). For siRNA-mediated depletion of PRPF8, Cal51 cells were transfected with DharmaFECT1 (Dharmafect) with 25 nM siRNA against human PRPF8. Transfected cells were harvested 54 hrs later, exposed to UV-C light and used for iCLIP. For collection of samples from different stages of the cell cycle, Cal51 cells were synchronized in G1/S by standard double thymidine block. Briefly, cells were treated with 1.5 mM thymidine for 8 hrs, washed and released for 8 hrs, then treated again with thymidine for a further 8 hrs. Cells were also collected 3 hrs (S-phase) and 7 hrs (G2) after release from the thymidine block. The iCLIP protocol was followed to isolate RNA bound to the spliceosome and amplify a cDNA library as described in the manuscript. An anti-SmB antibody was used for immunoprecipitation. Specific barcodes were used for each RT reaction, with random nucleotides also incorporated into cDNAs to distinguish between PCR duplication of cDNA products.
Experimental Factor: RNA interference
none
Experimental Factor: cell cycle phase
S phase
Experimental Factor: immunoprecipitate
anti-SmB