GSM8085829: Actg1 NSD native RIP rep 2; Mus musculus; RIP-Seq
native RIP was performed using the Magna Nuclear RIP (Native) Nuclear RNA-Binding Protein Immunoprecipitation Kit (Millipore Sigma) according to the manufacturer's protocol using at least 2x 107 WT or rescued Actg1-NSD MEFs per replicate. Enriched 'input' samples were generated by reserving 10% of the starting lysate; the remaining volume was subjected to IP using the with 10 µg Anti-ILF3 antibody (abcam; ab92355 and BD Biosciences; Clone 21/DRBP76) coated onto protein A/G magnetic beads as described in the Magna RIP technical manual. RNA purified from both IP and input samples was concentrated by ethanol precipitation and resuspended in equivalent volumes of RNase-free water to be used to generate RNA-seq libraries. 10ng of RNA was used to generate small RNA-seq libraries using the SMARTer smRNA-Seq Kit (Clonetech). RNA was treated with T4 Polynucleotide Kinase for 1 hr prior to library preparation to capture various potential mRNA decay intermediates that may not have a 5'P or 3'OH.