RNA immunoprecipitation and sequencing of ILF2-bound transcripts in Multiple Myeloma

To understand the mechanistic basis of ILF2’s regulation of mRNA splicing in response to DNA damage in Multiple Myeloma, we performed RNA immunoprecipitation (RIP) and sequencing of ILF2-bound transcripts under both physiological and DNA damage (melphalan treatment) conditions. Cells were treated with melphalan for 10 hours. RNA immunopreciptation (RIP) and sequencing of ILF2-bound RNAs was performed in the JJN3 and H929 cell lines (two biological replicates/condition). Cells were treated with melphalan for 10 hours. RNA immunoprecipitation (RIP) analyses were performed on untreated or melphalan treated JJN3 and H929 cells using anti-ILF2 (Abcam, ab113205) or anti-YB-1 (Abcam, ab12148) antibodies. In brief, cells were cross-linked in 0.1% formaldehyde for 10 min prior to harvest and lysis. RIP was performed using the Magna RIP RNA-binding protein immunoprecipitation kit (Millipore) according to the manufacturer’s instructions. RNA in each eluted sample was sequenced. Libraries were constructed using the Ovation RNA-Seq System V2 (Nugen) according to the manufacturer’s instructions. Reads were mapped to the human genome (GRCh38) using STAR . Isoform abundances were quantified using Cufflinks (version 2.2.1) . Low-abundance isoforms in the immunoprecipated samples (Fragments Per Kilobase of transcript per Million mapped reads [FPKM]<3) were excluded. Isoforms with significantly increased ILF-2 or YB-1 binding were identified using a q value <0.05 and a fold change >1.5 as cutoffs between the input and immunoprecipitate of each sample.