GSM7047197: Background_75-100kDa_Rep2; Homo sapiens; RIP-Seq
Protein-G beads (Thermo Fisher, 10004D) were washed twice in 1ml iCLIP Lysis buffer and resuspended in 100 µl per condition (100 µl of protein-G beads bind 20-30 µg of antibody - scale accordingly). Per IP, 1 µg of antibody against hnRNPc (Santa Cruz Biotechnology, sc-32308), RBM9 (Bethyl Laboratories, A300-864A), hnRNPA1 (4B10) (Santa Cruz Biotechnology, sc-32301), or hnRNPI (Santa Cruz Biotechnology, sc-56701) were added, and antibody-bead mixture was incubated at room temperature (RT) for 30-60 minutes on a rotating wheel. Antibody-bead mixture was washed twice in iCLIP lysis buffer to remove unbound antibody and RNase-treated cell lysates were added alongside cOmplete EDTA-free Protease Inhibitor Cocktail (Merck, #11836170001) and incubated for 2 hours at 4ºC on a rotating wheel. After IP, beads were washed twice in 200 µl 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), with the second wash at 4ºC for 3 minutes on a rotating wheel, followed by two washes in 200 µl PNK Wash Buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.2% Tween-20). Dephosphorylation of 3' ends was performed in 20 µl of PNK reaction for 30 minutes at 37ºC (70 mM Tris-HCl, pH 6.5, 10 mM MgCl2, 1 mM DTT, 10U SUPERaseIN RNase Inhibitor (ThermoFisher, #AM2696), 5U T4 Polynucleotide Kinase (NEB, #M0201L). Beads were washed twice in PNK Wash Buffer and resuspended in 20 µl of ligation mix for overnight incubation at 16ºC and 1200 rpm (50 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 1mM DTT, 10U SUPERaseIN RNase Inhibitor, 10U T4 RNA Ligase (NEB, #M0204), 1 µl of 1 µM L3 adapter and 20% PEG400 (Sigma-Aldrich, #91893)). Following the first adapter ligation, beads were washed twice in 200 µl High Salt Buffer, twice in 200 µl PNK Wash buffer and then resuspended in 20 µl 1X LDS sample buffer (Thermo Fisher, #NP0008) containing 5% beta-mercaptoethanol (Sigma-Aldrich, #M6250). Samples were denatured for 1 minute at 70ºC and RNA-protein complexes were resolved on NuPAGE 4-12% Bis-Tris Gels (Thermo Fisher, #WG1402A) at 180V for 1 hour. Transfer was performed onto nitrocellulose (BioRad, #1620115) in 1X NuPAGE transfer buffer (Thermo Fisher, #NP00061) with 10% methanol at 30V for 2 hours at RT.   Nitrocellulose membranes were scanned on Odyssey® CLx Infrared Imager (LI-COR, 9141) with 169 µm resolution to visualise RNA localisation and then placed on filter paper soaked in PBS. Regions of interest were cut out from nitrocellulose membrane corresponding to ~20-100 kDa above the molecular weight of the RBP of interest due to the ligation of L3 adapter (~15.9 kDa) and associated RNA (with 70nt of RNA averaging ~20kDa). Nitrocellulose pieces were placed in LoBind Eppendorf tubes and 200 µl Proteinase K buffer (100mM Tris-HCl, pH 7.4, 50 mM LiCl, 1 mM EDTA, 0.2% LiDS) containing 200 µg Proteinase K (Thermo Fisher, #AM2546) were added and incubated at 50ºC for 45 minutes at 800rpm.  Meanwhile, 10 µl of Oligo(dT)25 Dynabeads™ (Thermo Fisher, #61005) per sample were washed in 1 ml of oligo(dT) Binding Buffer (20 mM Tris-HCl, pH 7.4, 1 M LiCl, 2 mM EDTA) and resuspended in 50 µl of oligo(dT) Binding Buffer per sample. Following Proteinase K treatment, supernatant was transferred to fresh tubes containing 50 µl of oligo(dT) beads and incubated for 10 minutes at RT on a rotating wheel. Following RNA capture, beads were washed twice in 125 µl oligo(dT) Wash Buffer (10 mM Tris-HCl, pH 7.4, 150 mM LiCl, 0.1 mM EDTA) and once in 20 µl 1X First-Strand Buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2). Beads were resuspended in 10 µl of Reverse Transcription Mix (1X First-Strand Buffer, 0.5 mM dNTPs, 1 mM DTT, 6U SUPERase IN RNase Inhibitor, 20U SuperScript™ IV Reverse Transcriptase (Thermo Fisher, #18090050) and incubated for 15 minutes at 50ºC followed by 10 minutes at 80ºC heating up to 96ºC. Samples were vortexed for 30 seconds at 96ºC and then immediately placed on a magnet on ice. Supernatant containing adapter-ligated RNA was removed and efficiency of elution can be confirmed by dot-blotting on nitrocellulose membrane.   Solid-phase cDNA was washed once in 60 µl oligo(dT) Wash Buffer and once in 20 µl 1X T4 RNA Ligase Buffer (50 mM Tris-HCl, 10 mM MgCl2, 1mM DTT, pH 7.5). Beads were resuspended in 5 µl of 5' Adapter mix (2 µl 10X T4 RNA Ligase Buffer, 2 µl of 10 µM L## oligo (see Supplementary Table 2), 1 µl 100% DMSO), incubated at 75ºC for 2 minutes then immediately placed on ice. 4 µl of Ligation Mix (5 mM ATP, 7U Terminal Deoxynucleotidyl Transferase (TdT) (2230B, Takara), 15 U T4 RNA Ligase High Concentration (M0437, NEB)) were added as well as 10 µl 50% PEG8000 and reaction was mixed by pipetting up- and down until beads are resuspended. Reaction was incubated at 37ºC for 30 minutes, then cooled down to room temperature. 30 U of T4 RNA Ligase were added, the reaction mixed by pipetting and incubated at RT overnight with occasional vortexing for 15 seconds at 2000 rpm every two minutes.  Following overnight incubation, ligation reaction was removed and beads washed in 100 µl oligo(dT) Wash Buffer and 20 µl 1X Phusion HF Buffer (Thermo Fisher, #F518L). Beads were resuspended in 25 µl cDNA amplification mix (1X Phusion HF PCR Master Mix (NEB, #M0531L) and 0.5uM P5 and P7 short primer mix (see Supplementary Table 2)) and amplification was performed with the following programme: 30 seconds at 98ºC, 7 cycles of 10 seconds at 98ºC, 30 seconds at 65ºC and 30 seconds at 72ºC followed by final extension at 72ºC for 3 minutes. Meanwhile, 2µl of oligo(dT) beads per sample were washed once in 1 ml oligo(dT) Binding buffer and resuspended in 5 µl per sample. After cDNA amplification, 5 µl of oligo(dT) beads were added and incubated at RT for 5 minutes on a rotating wheel to capture unwanted amplification by-products (see Fig. S3). Samples were placed on magnet and supernatant containing amplified cDNA was transferred to a fresh tube.   Size-selection of cDNA was performed using ProNEX® Size-Selective Purification System (Promega, #NG2002) with a ratio of 2.8X to enrich for cDNA inserts of at least 20 nucleotides in length (>80bp). Library yield was then estimated by amplifying 1 µl of purified cDNA via qPCR using the full length P5 and P7 index primers and 2-3 cycles were subtracted from the obtained Ct value for final library amplification. Following PCR amplification, libraries were size-selected again using the ProNEX® Size-Selective Purification System, with a ratio of 1.8X to select fragments larger than 165bp. CLIP-seq