The RNA-protein complexes imaged as described above appeared as a diffused signal with a modal size of ∼15-20kDa above the expected MW of the protein of interest. Average MW of 21 nt long RNA is ∼7kDa. Poly(A) tail ∼20nt (∼6.5kDa), therefore the position of the protein-RNA complex that will generate CLIP tags longer than 20nt is ∼14kDa above the expected MW of the protein. HNRNPA2-Flag and HNRNPB1-Flag run at 38 and 39 kDa respectively and RBM24-Flag runs at ∼28. Therefore, we cut between 55 and 85 kDa for HNRNPA2 and HNRNPB1 lanes and 39–70 kDa for RBM24 lane. The MDA only (no flag) lane was cut from 39 to 85 kDa.
The cut membranes were each transferred to a 1.5 mL Eppendorf tube and treated with 12.5 μl Proteinase K in 200 μl Proteinase K digestion buffer at 55C for 45 min. The samples were then quickly spun down and the 200 μl of supernatant was transferred to a clean Eppendorf tube. Samples were then adjusted for salt by adding 19 μl 5M NaCl and 11 μl H2O per 200 μl sample.
To capture the RNA, we used 30 μl Oligo d(T)25 dynabeads (Invitrogen cat#61002) per IP. Beads were washed 2X with Proteinase K buffer before use. We transferred ∼200ul salt-adjusted samples to the beads and incubated at 25C at 300 RPM for 20 min with occasional shaking of 1350 RPM. We then washed the samples/beads 2X with cold high salt wash buffer and 2X with PBS, magnetized and removed the supernatant. RNA was eluted by incubating the beads in 8 μl TE elution buffer at 50C for 5 min. Beads were magnetized and 7.5 μl of eluted RNA was transferred to clean PCR tubes.
The cut membranes were each transferred to a 1.5 mL Eppendorf tube and treated with 12.5 μl Proteinase K in 200 μl Proteinase K digestion buffer at 55C for 45 min. The samples were then quickly spun down and the 200 μl of supernatant was transferred to a clean Eppendorf tube. Samples were then adjusted for salt by adding 19 μl 5M NaCl and 11 μl H2O per 200 μl sample.
To capture the RNA, we used 30 μl Oligo d(T)25 dynabeads (Invitrogen cat#61002) per IP. Beads were washed 2X with Proteinase K buffer before use. We transferred ∼200ul salt-adjusted samples to the beads and incubated at 25C at 300 RPM for 20 min with occasional shaking of 1350 RPM. We then washed the samples/beads 2X with cold high salt wash buffer and 2X with PBS, magnetized and removed the supernatant. RNA was eluted by incubating the beads in 8 μl TE elution buffer at 50C for 5 min. Beads were magnetized and 7.5 μl of eluted RNA was transferred to clean PCR tubes.
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