title: GSM1286117: FLAG-HA ZFP36; Homo sapiens; OTHER
library_construction_protocol: Cell pellets were lysed in NP40 lysis buffer, centrifuged, and the resulting supernatants used in subsequent steps. The cleared supernatants were incubated with Rnase T1 (Fermentas) at  22ºC, for 15 min, then cooled on ice. FLAG-antibody conjugate paramagnetic beads (Dynabeads, Invitrogen) were added to each supernatant to begin immunoprecipitation. Immunoprecipitates were subjected to dephosphorylation using calf-intestinal alkaline phosphatase (NEB), followed by three buffer washes, then treatment with T4 polynucleotide kinase (NEB) using gamma radiolabelled - 32P ATP. Radiolaballed immunoprecipitates were then fractionated by SDS-PAGE. The immunoprecipitated bands of interest were excised, electroeluted, and treated with Proteinase K to isolate crosslinked RNAs. Crosslinked RNAs were taken through a standard adapter-mediated small RNA cloning protocol and sequenced using Illumina GAII platform.
attributes.tag: GEO Accession
attributes.value: GSM1286117