GSM2973982: Trm3_pulldown_B; Saccharomyces cerevisiae; RIP-Seq
Cells were resuspened in 1.5 mL iCLIP lysis buffer (50 mM Tris-HCl pH7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1X protease inhibitor cocktail (Millipore), 400U/mL Murine RNase inhibitor (NEB)) per g of cell pellet and lysed by vortexing with glass beads (Manufacturer). Lysates were clarified by centrifugation, flash frozen in liquid N2 and stored at -80°C. Lysates were diluted in iCLIP lysis buffer to 26 ODU/2 mL and treated with Turbo DNase (Lifetech) and RNase I (Lifetech) for 15min at 22°C before placing on ice. Treated lysates were centrifuged (15,000 x g 15min) and added to Protein G magnetic beads (Dynabeads) pre-bound with 10 µg of antibody (anti-Myc or anti-FLAG, Sigma) and rotated for 2.5hr at 4°C. 4% of the binding reaction was saved as input before extensive washing of bead-bound protein-RNA complexes. Bound RNA 3′ ends were dephosphorylated with FastAP (Lifetech) and PNK (NEB) before on-bead ligation of a 3′ RNA linker with T4 Rnl (NEB) at 16°C overnight. After washing, 10% of bound RNA was labeled by PNK with 32P ATP for diagnostic gels. The remainder was eluted from the beads by incubation in 1X NuPAGE loading buffer with 0.1M DTT at 70°C for 10min. CLIP eluates and paired inputs were electrophoresed on 4-12% Bis-Tris NuPAGE and transferred to nitrocellulose membranes. Tagged protein MW was determined by western blot of 2% input before CLIP and size-matched input (SMI) membranes were cut, taking a 75kDa region just above the target protein size. RNA was released with proteinase K, extracted with phenol:chloroform:isoamy alcohol, and purified on RNA Clean & Concentrator-5 columns (Zymo). SMI RNA was dephosphorylated, a 3′ linker ligated, and purified on silane beads (Dynabeads). CLIP and SMI RNA was reverse transcribed with SuperScript IV, hydrolyzed with NaOH, neutralized with HCl, and cDNA was purified on silane beads. A 5′ linker was ligated with T4 Rnl (NEB) overnight at 16°C. Linker-ligated cDNA was purified on silane beads before diagnostic PCRs to determine the minimum number of cycles. Final PCR products were gel-purified, precipitated and sequenced on an Illumina HiSeq 2000.
GEO
GSM2973982