GSM2905631: DM.2 [RISC-IP long RNA]; Homo sapiens; RIP-Seq
Two lysis protocols were used: 1) skeletal muscle biopsies were cut into small pieces in RIP lysis buffer (Magna RIP Kit, Merck-Millipore, Burlington, MA) and homogenized by TissueLyser (Qiagen, Hilden, Germany); 2) Cultured cells were lysed with the following buffer: 150mM KCl, 25 mM TrisHCl pH 7.4, 5 mM EDTA, 0.5% NP40, 5 mM DTT, 1 mM PMSF, protease and RNase inhibitors. For both lysates, homogenates were quantified for protein content, snap frozen in liquid nitrogen and stored at -80°C until use. For both muscle tissue and cells, 1 mg of lysate was immunoprecipitated with anti-Ago2 or control mouse IgG antibodies (RIPAb+ Ago2, Merck-Millipore, Burlington, MA), using 5 ug of antibody for 1 mg of lysate. Immunoprecipitation was performed using the Magna RIP Kit (Merck Millipore, Burlington, MA) following the manufacturer's instructions. IgG-bound RNA (IP RNA) was extracted from magnetic beads using TRIzol reagent (Invitrogen, ThermoFisher Scientific, Waltham, MA). Extraction, ribodepletion and library preparation were performed following manufacturer's instructions for the Nugen OVATION RNA-Seq System V2 and Nugen OVATION Ultralow Library Systems.
GEO
GSM2905631
GEO Accession
GSM2905631