GSM2497682: NF90+NF110 KO RIP-seq IP rep3; Mus musculus; RIP-Seq
Total RNA was collected using TRIzol (ThermoFisher Scientific) according to the manufacturer’s protocol. For NF45 WT and NF45 KO ESCs, 1ug of RNA per sample was processed using the Illumina TruSeq Stranded mRNA Sample Prep kit according to the manufacturer’s protocol. For NF90/NF110 WT, NF90+NF110 KO, and NF110 KO ESCs, 1ug of RNA per sample was processed using the KAPA Stranded RNA-Seq with RiboErase kit according to the manufacturer’s protocol. RIP was performed essentially as previously described. ESCs were harvested, pelleted, and lysed in 10 pellet volumes of polysome lysis buffer (100mM KCl, 5mM MgCl2, 10mM HEPES pH 7.5, 0.5% NP40 substitute, 1mM DTT) with cOmplete protease inhibitors (Roche), 100U/ml Superase-In (ThermoFisher Scientific), and 400uM vanadyl ribonucleoside complex (NEB). Lysates were freeze-thawed once, incubated with 100U/ml Turbo DNase (ThermoFisher Scientific) for 15min at 37C, and then cleared by centrifugation at maximum speed for 15min at 4C. Aliquots of the cleared lysate were taken at this step to serve as input for Western blot and qRT-PCR analysis. Antibodies for immunoprecipitation (5ug NF90/NF110 Ab/1ml lysate [BD Biosciences clone 21/DRBP76], 6ul normal rabbit IgG/1ml lysate [ThermoFisher Scientific]) were conjugated to 40ul/1ml lysate Protein G Dynabeads (ThermoFisher Scientific) in polysome lysis buffer for 2 hours at room temperature. Lysates were then incubated with Protein G Dynabead-conjugated antibodies for 4hr at 4C. Immunoprecipitates were washed 3x with polysome lysis buffer and then 3x with polysome lysis buffer containing 1M urea. An aliquot of the immunoprecipitate was taken for Western blot, while the rest of the immunoprecipitate, along with the qRT-PCR input lysate, was incubated with 1.2mg/ml proteinase K (Roche) and 2x proteinase K buffer (100mM Tris-HCl pH7.5, 12.5mM EDTA, 150mM NaCl, 2% SDS) for 30min at 55C. Samples were then lysed in TRIzol LS (ThermoFisher Scientific), and RNA was extracted and used for qRT-PCR or sequencing.
GEO Accession
GSM2497682