title: GSM3611422: imb_koenig_2014_03_16_JKRS25_U2AF65Nx_1; Homo sapiens; RIP-Seq
library_construction_protocol: The in vitro iCLIP protocol was developed by modifying the early steps of the standard iCLIP protocol (Huppertz et al., 2014; Sutandy et al., 2016). Briefly, beads were prepared by twice washing 40 µl of protein-G Dynabeads per sample with dilution buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate; corresponding to the lysis buffer in the in vivo iCLIP protocol). After the second wash, 40 µl of dilution buffer was added to resuspend the beads and then followed by mixing with 3 µg of anti-U2AF65 antibody. The beads were rotated at room temperature for 30-60 min. One-time high-salt buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate) and twice dilution buffer washes were applied to wash the beads before proceeding with immunoprecipitation.  The in vitro transcripts were preheated for 5 min at 70°C to reduce large-scale RNA secondary structures. Titrated concentrations (30 nM, 50 nM, 150 nM, 250 nM, 450 nM, 750 nM, 1.5 µM, 3 µM, 5 µM, 15 µM) of U2AF65RRM12 and 2.2 nM in vitro transcript mix (eleven transcripts) were used for the Kd measurements. For the initial hnRNPC1 titration experiment, 1 µM U2AF65RRM12 was mixed with 6.75 nM in vitro transcript mix (nine transcripts; excluding MALAT1 and MIRLET7A2) and different concentrations of recombinant hnRNPC1 (200 nM, 500 nM, and 1 µM) in binding buffer. For the co-factor experiments, 500 nM U2AF65RRM12 was mixed with 6.75 nM in vitro transcript mix (nine transcripts) and different concentrations of eleven recombinant RBPs in binding buffer. In addition, 500 nM BSA was added to 500 nM U2AF65RRM12 and 6.75 nM in vitro transcript mix as a control for in vitro iCLIP co-factor assays.    All in vitro mixtures were incubated for 10 min at 37°C. After the incubation, the mixtures were placed on top of a parafilm-coated plate and UV-irradiated with 5 mJ/cm2 250 nm UV wavelength (Stratalinker 2400). The irradiated in vitro mixtures were pooled back to the tubes, and dilution buffer was added to fill the samples to a volume of 1 ml. To normalize the final in vitro iCLIP libraries, 10 µl of crosslinked mixture containing 250 nM U2AF65RRM12 and 6 nM NUP133 in vitro transcript was spiked in to each sample. Partial RNase digestion was performed by adding 10 µl of 1:1500 diluted RNase I (Ambion) to each sample. In addition, 2 µl of TURBO DNase was added to each sample to avoid DNA contamination. The sample mixtures were incubated for 3 min at 37°C, added to the prepared beads, and incubated for 2 h at 4°C. Beads were washed twice with high-salt buffer and twice with wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% Tween-20).   Henceforth, we followed the steps of the standard iCLIP protocol. Briefly, 3' end RNA dephosphorylation was performed by resuspending the beads in 20 µl of mixture containing 4 µl of 5x PNK buffer (350 mM Tris-HCl pH 6.5, 50 mM MgCl2, 5 mM DTT), 0.5 µl of PNK (NEB), 0.5 µl RNasin Ribonuclease Inhibitor (Promega), and 15 µl water, followed by incubation for 20 min at 37°C. The beads were washed once with wash buffer, once with high-salt buffer and twice with wash buffer.  For the linker ligation, pre-adenylated L3 linker (5'-App-AGATCGGAAGAGCGGTTCAG-dideoxycytidine-3') was ligated by resuspending the beads in the ligation mixture containing 5 µl 4x ligation buffer (200 mM Tris-HCl pH 7.8, 40 mM MgCl2, 4 mM DTT), 1 µl T4 RNA ligase (NEB), 0.5 µl RNasin, 1.5 µl pre-adenylated L3 linker (20 µM), 4 µl PEG400, and 8 µl water. The samples were incubated at 16°C overnight. The next day, the samples were washed twice with high-salt buffer and twice with wash buffer.   Interacting RNAs were radioactively labeled by resuspending the beads in hot PNK mix (0.2 µl PNK [NEB], 0.4 µl 10x PNK buffer [NEB], 0.4 µl 32P-γ-ATP, and 3 µl water). The beads were incubated at 1,100 rpm for 5 min at 37°C. Supernatants were removed and the beads were boiled in 20 µl 1x NuPAGE loading buffer (Invitrogen) for 5 min at 70°C. Boiled beads were placed on a magnetic rack. The supernatants were then loaded into the 4-12% NuPAGE Bis-Tris gel (Invitrogen) and run in 1x MOPS buffer for 50 min at 180 V. Protein-RNA complexes from the gel were transferred to a nitrocellulose membrane for 1 h at 30 V.  To extract the interacting RNAs, the membrane was cut into pieces and digested with 10 µl proteinase K (Roche) in 200 µl PK buffer (100 mM Tris-HCl pH 7.4, 50 mM NaCl, 10 mM EDTA) for 20 min at 37°C. Another 200 µl PK buffer containing 7 M urea were added for further 20 min incubation at 37°C. The RNA-containing mixtures were transferred to Phase Lock Gel Heavy tubes and mixed with 400 µl phenol/chloroform by shaking with 1,100 rpm for 5 min at 30°C. RNAs were extracted by centrifugation for 5 min at 16,000 xg to separate the phases followed by transferring the top aqueous phase containing RNAs to new tubes. The samples were then mixed with 0.75 µl glycoblue (Ambion), 40 µl 3 M sodium acetate pH 5.5 and 1 ml ethanol absolute, and incubated overnight at -20°C. To precipitate the RNAs, the samples were centrifuged with 21,000 xg for 20 min at 4°C, washed with 80% ethanol, and resuspended in 5 µl water. cDNA synthesis was performed by adding 1 µl dNTP mix and 1 µl RT primers containing different barcode sequences to each sample, and incubating them for 5 min at 70°C. The reaction was started by adding RT mixture (4 µl 5x RT buffer [Invitrogen], 1 µl 0.1 M DTT, 0.5 µl RNasin, 0.5 µl Superscript III [Invitrogen], 7 µl water) to the samples and incubating them for 5 min at 25°C, 20 min at 42°C, 40 min at 50°C, 5 min at 80°C, and hold at 4°C. To hydrolyze hot RNA templates, 1.65 µl 1 M NaOH was added, followed by 20 min incubation at 98°C. After the incubation, 20 µl 1 M Hepes-NaOH was added to neutralize the samples' pH. The cDNA libraries were mixed with 0.75 µl glycoblue, 40 µl 3 M sodium acetate pH 5.5 and 1 ml ethanol absolute, and incubated overnight at -20°C. The next day, the cDNA libraries were precipitated by spinning the samples with 21,000 xg for 20 min at 4°C, washing with 80% ethanol, and resuspension in 6 µl water.   cDNA libraries were mixed with 6 µl 2x TBE-urea loading buffer (Invitrogen), heated for 5 min at 80°C, and then loaded and run in a 6% TBE-urea gel for 40 min at 180 V. DNA low molecular weight size marker (NEB) was used as the ladder. The libraries were size-selected by cutting out the gel within the range of 80-100 nt based on the ladder. Each piece of the gel was then crushed into smaller pieces and mixed with 400 µl diffusion buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, 0.1% SDS). The mixtures were incubated for 30 min at 50°C, and moved to a Costar SpinX column (Corning) prepared with two 1 cm glass pre-filters (Whatman). To extract the cDNA libraries, the mixtures were spun at 16,000 xg for 5 min, and the eluates were added together with 400 µl phenol/chloroform into a Phase Lock Gel Heavy tube. The samples were incubated for 5 min at 30°C, and spun at 16,000 xg for 5 min to separate the phases. The aqueous top layers containing the libraries were moved to new tubes, mixed with 1 µl glycoblue, 40 µl 3 M sodium acetate pH 5.5 and 1 ml ethanol absolute, and then stored at -20°C overnight.  For circularization, libraries were centrifuged with 21,000 xg for 20 min at 4°C, washed with 80% ethanol, and resuspended in 8 µl of ligation mixture (0.8 µl 10x CircLigase buffer II [Epicentre], 0.4 µl 50 mM MnCl2, 0.3 µl CircLigase II [Epicentre], and 6.5 µl water). The libraries were transferred into PCR tubes and incubated for 1 h at 60°C. To re-linearize the libraries, 30 µl oligo annealing mix containing 3 µl FastDigest buffer (Thermo Fischer), 1 µl 10 µM cut_oligo (5'-GTTCAGGATCCACGACGACGACGCTCTTCaaaa-3'), and 26 µl water were added. The annealing program was performed by running the samples in succ
attributes.tag: GEO Accession
attributes.value: GSM3611422