GSM7047191: noUV_noPAGE_RBFOX2_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®