GSM8078191: RBM42 Cytoplasmic CLIP bio rep 2; Homo sapiens; RIP-Seq
Protein-bound RNAs were dephosphorylated on bead using T4 PNK (NEB) with modified PNK Buffer (10mM Tris pH 6.5, 50mM MgCl2, 50mM DTT) followed by PolyA tailing with yeast poly(A) polymerase (Jena). This was followed by 3′-end labelled with azide-dUTP, using yeast poly(A) polymerase (Jena) and labelled with IRDye800-DBCO conjugates (LiCor). The protein–RNA complexes were then eluted from beads, resolved on a 4–15% gel, transferred to nitrocellulose and imaged using an Odyssey CLx instrument (LiCor). Regions of interest were excised from the membrane, and the RNA was isolated by Proteinase K digestion and oligo-dT dynabead purified in high salt (500mM NaCl) buffer to promote capture. RNA was eluted into TE buffer (20mM Tris pH 7.5, 1mM EDTA). Library preparation using SMARTer smRNA-Seq Kit (Takara) with custom primer for reverse transcription (custom RT primer:CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTT, Ns = UMI).