GSM1167167: RBM5 CLIP-Seq Replicate 2; Homo sapiens; OTHER
HeLa cells were grown in 150mm plates, rinsed once with ice-cold PBS and placed in a Stratalinker without the cover plate and irradiated once with an energy of 400mJ/cm. Cells were then scraped off the palte and centrifuged at 2500rpm for 5 min at 4°C and the pellet re-suspended in 3x the cells volume of PBS and distributed in two separate eppendorf tubes. Each tube of crosslinked lysate was resuspended in 700 μl of 1X PXL (PBS (tissue culture grade without Mg++ or Ca++) 0.1% SDS 0.5% deoxycholate 0.5% NP-40, supplemented with protease inhibitors and with 15 μl RNAsin (Promega)) and kept on ice for 10 min. 30 μl of RQ1 DNAse (Promega, M6101) were added to each tube and extracts were incubated at 37° for 5 min, centrifuged at 1000 rpm, followed by an incubation at 37° for 5 minutes with RNAse I. Extracts were the centrifuged at 12000 rpm for 10’ and then clarified on protein A Dynabeads  for 2 hours at 4°C,  before immunoprecipitation using protein A pre-adsorbed with either rabbit IgGs or EWS antibody (5μg) at 4 °C under constant rotation. After incubation for 3 hours, stringent washes were performed with ice-cold buffers as follows: 2 washes with 1X PXL, 2 washes with 5X PXL (High-salt Wash Buffer) and 2 washes with PNK Buffer (50 mM Tris-Cl pH 7.4 10 mM MgCl2 0.5% NP-40). Alkaline phosphatase (Roche) treatment (3μl) was performed at 37°C for 20’ in Thermomixer, followed by 4 washes in PNK buffer (one wash including 20mM EGTA). 3’ RNA linker ligation was carried out overnight at 16°C on beads using T4 RNA ligase (Fermentas) and radioactively-labeled linkers. Beads were extensively washed (1x PXL buffer, 1x 5XPXL buffer, 3x PNK buffer), before and after PNK treatment (T4, NEB, [40 units]) for 20’ at 37°C.  Finally, beads were resuspended in 30ml of PNK buffer, supplemeted with Novex loading buffer and loaded on a Novex NuPAGE 10% Bis-Tris gel. The gel was run at 175V in the cold room and subsequently transfered to nitrocellulose using the Novex wet transfer apparatus, for 1 h at 30V in NuPAGE Transfer Buffer with 10% methanol. After transfer, the nitrocellulose filter was rinsed in 1X PBS, and exposed to autoradiogram. The radioactively labeled RNA-protein complexes were excised and proteinase K treatment was performed (20’ at 37°C) before RNA extraction with Phenol-Chloroform and preciiptation overnight with 3M NaOAc pH 5.2, glycogen (Ambion, 9510) and 1:1 EtOH:isopropanol. The RNA was resuspended in RNase free water and 5’ end linker ligation (containing also barcode sequences) was carried out with T4 RNA ligase at 16°C for 5 hours. RNAs were reverse-transcribed, PCR amplified with barcoded primers and sequenced using Solexa (Illumina) technology.