GSM8147167: SHSY5Y WT Cyto rep 2 (AGO); Homo sapiens; RIP-Seq
Cells were fractinated into cytoplasmic and nuclear lysates and clarified by centrifugation. First AGO / FAM120A proteins were enriched by immunoprecipitation with T6B peptide or anti-FAM120A antibody. After washing the beads, RNase T1 digestion was performed, 3´ends of the RNA fragments were dephosphorylated using CIP, and RNA was fluorescently 3´end-labeled using T4 Rnl2(1–249)K227Q, followed by dephosporylation with T4 PNK. RNA-protein complexes were separated by SDS-PAGE and excised. After proteinase K treatment, RNA was extracted and 5' end labelled with T4 RNA ligase. Following small RNA cDNA library preparation was performed. Ligation products were reverse transcribed and amplified by PCR.