GSM8085860: WT crosslinked input rep2; Mus musculus; RIP-Seq
Cross-linked RIP was performed using the Magna Nuclear RIP (Cross-Linked) 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. For the cross-linked RIP, fixed nuclei were subjected to sonication using Bioruptor (Diagenode) to generate fragments of 200–600 bp in size prior to IP. 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 sequencing libraries were generated using 10 ng of RNA from input and IP samples using the SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Clontech).