GSM1568713: hDIS3_PAR-CLIP; Homo sapiens; RIP-Seq
Cells were scraped off and centrifuged at 400 x g for 5 min. at 4°C. 5 plates yielded 1 ml of wet cell pellet, which was sufficient for one experiment. Cell pellets were resuspended in lysis buffer (Expanded View Text), sonicated and centrifuged at 16000 x g for 15 min. at 4°C. To obtain RNA of an appropriate size, cleared extracts were treated with 5U/ml of RNAse T1 (Ambion) for 10 min. at 22°C with shaking at 150 rpm and cooled on ice for 5 min. Cell lysates containing RNA protein complexes were then mixed with urea at a final concentration of 0.5M and added to 50µl of freshly washed magnetic beads coated with GFPtrap antibodies and incubated for 1 hour at 4°C with rotation. Co-immunoprecypitation was followed by 3 washes with high salt wash buffer (HS buffer), 1 wash with low salt wash buffer (LS buffer) and 1 wash with PNK buffer (Expanded View Text). Beads resuspended in 50µl of PNK buffer were treated with 35U of alkaline phosphatase (NEB) in the presence of RNAse inhibitor (Ribolock; Thermo Scientific) for 20 min. at 37°C with shaking at 1100 rpm in Thermomixer (Eppendorf) and immediately washed 2 times with PNK buffer and 2 times with ligation buffer (Expanded View Text). The resulting short RNA fragments, dephosphorylated at their 3’ ends and still crosslinked to proteins, were ligated with a pre-adenylated linker Ra3 (Illumina). The reaction was doneby treating with 400U of T4 RNA Ligase 2, truncated (NEB) overnight at 16°C with shaking at 1100 rpm in 50µl of linker ligation buffer in the presence of 20% PEG 400 (Sigma) and 1mM DTT (Sigma). This step was followed by washing once with HS buffer, twice with LS buffer and once with PNK buffer. RNA molecules crosslinked to immunoprecipitated proteins were then radiolabeled at their 5’ ends. Beads were resuspended in 50µl of PNK buffer with 15U of T4 polynucleotide kinase (NEB; 10) and [32P]-γ-ATP (jak wpisać ilość izotopu?) and incubated at 37°C with shaking at 1100 rpm for 15 min. Then cold ATP was added and the incubation continued for 15 more minutes. Beads with radiolabeled RNA were then washed 4 times with PNK buffer, resuspended in NuPAGE LDS loading buffer (Life Technologies) and incubated for 10 min at 70°C with shaking 1100 rpm to denaturate and release RNAs-RBPs complexes. Then DTT was added to the final concentration of 65mM. RNAs-RBPs complexes were resolved by size using 4-12% NuPAGE Bis-Tris gel (Life Technologies) in MOPS SDS running buffer (Invitrogen) and transferred from the gel onto Protran Nitrocellulose membrane (Whatman) by wet-blotting (30V, 1h) in a Invitrogen XCell II Blot Module using NuPAGE transfer buffer (Invitrogen) according to the manufacturer's descriptions. The membrane was stained in with Ponceau S Red (Sigma-Aldrich; 0.1% in 3% acetic acid) and exposed to a phosphoscreen for 30 min. The RNA-RBPs complexes migrated above the expected MW of the protein, and appeared as a lengthy signal (Figure E1). The area of the membrane where radioactivity appeared was cut into small slices and placed in 400µl of PK buffer (Expanded View Text). RNAs were released by proteinase K digestion: Proteinase K (BioLine) was added to the samples at a final concentration of 500µg/ml and incubated at 37°C at 1100 rpm for 1h. The solution containing RNAs was then mixed with phenol/chloroform and shaken at 1100 rpm. Phases were separated by spinning for 5 min. at 16000 x g at room temperature. The aqueous layer was mixed with chloroform (1:1), shaken and centrifuged as described above. RNAs were precipitated overnight in the presence of Glycoblue (Ambion), 3M NaAc and ethanol. RNA molecules were then purified using magnetic beads (Agencourt AMPure XP; Beckman Coulter) according to the manufacturer's protocol. RNA library from the obtained RNA were prepard using ScriptSeq™ v2 RNA-Seq Library Preparation Kit (Epicentre) according to the manufacturer's instructions. cDNA synthesis primer, reverse PCR primer containing user-defined barcode and pre-adenylated linker Ra3 came from TruSeq Small RNA Sample Preparation Kit (Illumina).