title: GSM2724030: NAT10_ac4C_RIP; Homo sapiens; RIP-Seq
library_construction_protocol: Total RNA was purified from cultured cells using Trizol (ThermoFisher) followed by treatment with TurboTM DNase (ThermoFisher). Enrichment of polyadenylated RNA (poly(A)-RNA) from DNase-treated total RNA was carried out by two rounds of selection with Oligo-(dT)25 Dynabeads (ThermoFisher) according to the manufacturer’s instructions. Enrichment efficiencies were estimated with Bioanalyzer picoRNA chips (Agilent Technologies, Santa Clara, CA). Poly(A)-RNA from parental and NAT10-/- HeLa cells (pool of two clones) was fragmented using NEBNext® Magnesium RNA Fragmentation buffer for 5 min at 94 oC. 8 pg of ac4C-RNA probe was spiked into 8 mg of fragmented poly(A)-RNA (8 mg) followed by immunoprecipitation with 1 mg anti-ac4C or IgG pre-coupled Protein G Dynabeads Illumina libraries were constructed for inputs (parental vs NAT10-/-), acRIPs (parental vs NAT10-/-), and IgG (parental) using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®.
attributes.tag: GEO Accession
attributes.value: GSM2724030