SRX2884392 GSM2650326 SRP108637 SRS2252575 GSM2650326 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650326 GSM2650326 GEO Accession GSM2650326 SRX2884393 GSM2650327 SRP108637 SRS2252576 GSM2650327 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650327 GSM2650327 GEO Accession GSM2650327 SRX2884394 GSM2650328 SRP108637 SRS2252577 GSM2650328 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650328 GSM2650328 GEO Accession GSM2650328 SRX2884395 GSM2650329 SRP108637 SRS2252578 GSM2650329 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650329 GSM2650329 GEO Accession GSM2650329 SRX2884396 GSM2650330 SRP108637 SRS2252579 GSM2650330 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650330 GSM2650330 GEO Accession GSM2650330 SRX2884397 GSM2650331 SRP108637 SRS2252580 GSM2650331 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650331 GSM2650331 GEO Accession GSM2650331 SRX2884398 GSM2650332 SRP108637 SRS2252581 GSM2650332 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650332 GSM2650332 GEO Accession GSM2650332 SRX2884399 GSM2650333 SRP108637 SRS2252582 GSM2650333 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650333 GSM2650333 GEO Accession GSM2650333 SRX2884400 GSM2650334 SRP108637 SRS2252583 GSM2650334 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650334 GSM2650334 GEO Accession GSM2650334 SRX2884401 GSM2650335 SRP108637 SRS2252584 GSM2650335 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650335 GSM2650335 GEO Accession GSM2650335 SRX2884402 GSM2650336 SRP108637 SRS2252585 GSM2650336 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650336 GSM2650336 GEO Accession GSM2650336 SRX2884403 GSM2650337 SRP108637 SRS2252586 GSM2650337 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650337 GSM2650337 GEO Accession GSM2650337 SRX2884404 GSM2650338 SRP108637 SRS2252587 GSM2650338 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650338 GSM2650338 GEO Accession GSM2650338 SRX2884405 GSM2650339 SRP108637 SRS2252588 GSM2650339 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650339 GSM2650339 GEO Accession GSM2650339 SRX2884406 GSM2650340 SRP108637 SRS2252589 GSM2650340 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650340 GSM2650340 GEO Accession GSM2650340 SRX2884407 GSM2650341 SRP108637 SRS2252590 GSM2650341 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650341 GSM2650341 GEO Accession GSM2650341 SRX2884408 GSM2650342 SRP108637 SRS2252591 GSM2650342 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650342 GSM2650342 GEO Accession GSM2650342 SRX2884409 GSM2650343 SRP108637 SRS2252592 GSM2650343 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650343 GSM2650343 GEO Accession GSM2650343 SRX2884410 GSM2650344 SRP108637 SRS2252593 GSM2650344 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650344 GSM2650344 GEO Accession GSM2650344 SRX2884411 GSM2650345 SRP108637 SRS2252594 GSM2650345 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650345 GSM2650345 GEO Accession GSM2650345 SRX2884412 GSM2650346 SRP108637 SRS2252595 GSM2650346 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650346 GSM2650346 GEO Accession GSM2650346 SRX2884413 GSM2650347 SRP108637 SRS2252596 GSM2650347 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650347 GSM2650347 GEO Accession GSM2650347 SRX2884414 GSM2650348 SRP108637 SRS2252597 GSM2650348 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650348 GSM2650348 GEO Accession GSM2650348 SRX2884415 GSM2650349 SRP108637 SRS2252598 GSM2650349 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650349 GSM2650349 GEO Accession GSM2650349 SRX2884416 GSM2650350 SRP108637 SRS2252599 GSM2650350 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650350 GSM2650350 GEO Accession GSM2650350 SRX2884417 GSM2650351 SRP108637 SRS2252600 GSM2650351 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650351 GSM2650351 GEO Accession GSM2650351 SRX2884418 GSM2650352 SRP108637 SRS2252601 GSM2650352 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650352 GSM2650352 GEO Accession GSM2650352 SRX2884419 GSM2650353 SRP108637 SRS2252602 GSM2650353 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650353 GSM2650353 GEO Accession GSM2650353 SRX2884420 GSM2650354 SRP108637 SRS2252603 GSM2650354 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650354 GSM2650354 GEO Accession GSM2650354 SRX2884421 GSM2650355 SRP108637 SRS2252604 GSM2650355 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650355 GSM2650355 GEO Accession GSM2650355 SRX2884422 GSM2650356 SRP108637 SRS2252605 GSM2650356 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650356 GSM2650356 GEO Accession GSM2650356 SRX2884423 GSM2650357 SRP108637 SRS2252606 GSM2650357 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650357 GSM2650357 GEO Accession GSM2650357 SRX2884424 GSM2650358 SRP108637 SRS2252607 GSM2650358 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650358 GSM2650358 GEO Accession GSM2650358 SRX2884425 GSM2650359 SRP108637 SRS2252608 GSM2650359 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650359 GSM2650359 GEO Accession GSM2650359 SRX2884426 GSM2650360 SRP108637 SRS2252609 GSM2650360 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650360 GSM2650360 GEO Accession GSM2650360 SRX2884427 GSM2650361 SRP108637 SRS2252610 GSM2650361 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650361 GSM2650361 GEO Accession GSM2650361 SRX2884428 GSM2650362 SRP108637 SRS2252611 GSM2650362 RIP-Seq TRANSCRIPTOMIC other 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 Illumina MiSeq gds 302650362 GSM2650362 GEO Accession GSM2650362