title: GSM1915729: Transgenic Flag-FTO mice/IgG-IP 1; Mus musculus; RIP-Seq
library_construction_protocol: Transgenic FTO mice were fasted overnight and adipose tissues were lysed in passive lysis buffer (10 mM HEPES, pH 7.0, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40) containing 1 mM DTT, SUPERase In (100 U/mL, Ambion), and protease inhibitor cocktail (Roche). Homogenates were then gently lysed with repeated freezing at -80 °C and subsequent thawing. Passive lysis buffer lysates were diluted 10-fold in NET-2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40, 20 mM EDTA, pH 8.0, 1 mM DTT, 100 U/mL SUPERase In), precleared with protein-G beads, and immunoprecipitated with antibody specific to FLAG M2 agarose (Sigma-Aldrich, cat# F1804) or IgG for 90 min. Agaroses were washed 6 times with cold NT-2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1% NP-40). The input and beads were resuspended in 150 μL proteinase K buffer (NT-2 buffer supplemented with 1% SDS and 1.2 mg/mL proteinase K) for 30 min at 55 °C. Immunoprecipitated RNA was extracted by acid-phenol/chloroform for further analysis. Illumina HiSeq 2000 system.
attributes.tag: GEO Accession
attributes.value: GSM1915729