GSM8219020: CLIP-seq, HEK293T, DHX36-KO, Rep1; Homo sapiens; OTHER
Immunopreciptation was performed using 10 µg anti-YTHDF1 antibody, or normal IgG control respectively for overnight at 4°C with rotation. Next day, 125 µl washed Dynabeads Protein G was added to each reaction and incubated for another 4 hours with rotation. Beads were collected and washed twice with high salt wash buffer and twice with wash buffer. After washing, 1/10 volume of beads were saved to determine immunoprecipitation efficiency. The remaining cross-linked RNA complexes were dephosphorylated by incubating with Quick CIP for 20 min at 37°C, followed by treatment with T4 PNK enzyme in acid PNK buffer for 20 min at 37°C. The RNA-protein complexes were used for on-bead 3' RNA linker ligation and incubated in a 25 µl reaction volume. The beads were then denatured in loading buffer and resolved on a Novex NuPAGE 4-12% Bis-Tris gel and then transferred to 0.45 µm nitrocellulose membranes. The biotin reaction group was then subjected to biotin detection. For CLIP-seq, labeled YTHDF1-RNA bands were excised from the membrane and eluted. RNA was subsequently purified and 5' ends phosphorylated with T4 PNK and then ligated to 5' RNA adaptor. The resultant RNA was reverse transcribed with RP1 primer. The full-scale PCR amplification was performed.