GSM7916598: HEK293 PR8 infected nuclear lysate biol rep2; Homo sapiens; RNA-Seq
Cells were fractinated into cytoplasmic and nulcear lysates or lysed into total lysates and clarified by centrifugation. First AGO proteins were enriched by immunoprecipitation with T6B peptide. 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.