Illumina HiSeq 2000 sequencing; Transcriptome-wide mapping of RNA binding sites of the RNA-binding protein Unkempt using iCLIP-seq
Transcriptome-wide mapping of RNA binding sites of the RNA-binding protein Unkempt using iCLIP-seq
HeLa, and SH-SY5Y cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 micro-g/mL streptomycin at 37  degrees C and 5% CO2. SH-SY5Y and GFP and Unkempt-inducible HeLa cells were grown in 10 cm plates and harvested at 80% confluency. Prior to harvest, the inducible HeLa cells were treated with Dox for 24 hours to induce expression of Unkempt. iCLIP experiments on SH-SY5Y cells, HeLa cells, and mouse embryonic brain were carried out in replicates as described in Konig et al., JoVE, 2011. Whole brains of E15.5 CD1 mouse embryos were processed as described (Ule et al., Methods, 2005). Briefly, ten whole embryonic brains were isolated and transferred to 6 ml of PBS in a 15 ml tube on ice. Brains were triturated first through a 5 ml serological pipette and then through a 200 ?l pipette tip fitted to a 5 ml serological pipette to partially dissociate the tissue. The suspension was transferred to a 10 cm petri dish and irradiated with UV-light (254 nm) three times at 100 mJ/cm2 on ice, mixing suspension after each irradiation pulse.  The irradiated suspension was aliquoted into three 2 ml tubes and centrifuged at 2500 rpm for 3 minutes at 4  degrees C. The supernatant was removed and the cell pellets frozen at -80 degrees C until use. Immunoprecipitation of the cross-linked Unkempt-RNA complexes from all three sample types was carried out using anti-Unkempt antibody (HPA023636, Sigma-Aldrich). Control reactions were performed using normal rabbit IgG antibodies (sc-2027). Library preparation was performed as described in Konig et al, JoVE, 2011.
Experimental Factor: organism part
whole brain
Experimental Factor: genotype
wild type Unk
Experimental Factor: immunoprecipitate
anti-Unkempt antibody