title: Illumina Genome Analyzer IIx sequencing; Spliceosome iCLIP
design: Spliceosome iCLIP
library_construction_protocol: 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.
attributes.tag: Experimental Factor: RNA interference
attributes.value: none
attributes.tag: Experimental Factor: cell cycle phase
attributes.value: G1 phase
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: anti-SmB