GSM7505777: RIP_miRNA-M26A_ES_T_Cre_IgG_replicate 3; Mus musculus; RIP-Seq
For RIP-seq analysis, the lungs of infected mice were perfused with formaldehyde to form crosslinks between miRNAs, MREs and individual proteins of bound RNA induced silencing complexes (RISCs). Crosslinked miRNA-mRNA-RISC complexes were immunoprecipitated by the addition of an antibody against AGO2, one component of RISC, and magnetic beads conjugated to protein A . Additional samples received either an antibody against IgG, as a negative control, or no antibody (labeled as 10% input), as a positive control. Protein-RNA complexes isolated from each sample were washed and eluted from the magnetic beads, crosslinks were reversed and protein components were digested. For SIR analysis, lungs from late-stage pulmonary adenocarcinoma animals infected with either adenoCre or adeno were formaldehyde crosslinked. Crosslinked miRNA-MRE complexes were isolated by the addition of a SIR probe followed by streptavidin beads. After extensive washing, complexes were eluted, crosslinks were reversed and the liberated miRNAs were sequenced. As positive controls, sequencing libraries were also generated from 10% of the starting input from each sample. Sequencing libraries were constructed starting with the entire pellet of RIP or SIR RNA, using TruSeq Small RNA kits from Illumina (San Diego, CA), following manufacturer's instructions. Two different gel fragments were isolated from each gel of RIP samples. The first fragment, containing miRNAs, corresponded to a size of 145 bps-160 bps, including ligated adapters. The second fragment of each gel, containing immunoprecipitated mRNA targets, corresponded to a size of 160 bps-500 bps, including ligated adapters. Only one gel fragment, containing miRNAs corresponding to a size of 145 bps-160 bps, including ligated adapters, was isolated from each SIR sample. Libraries were checked for quality and concentration using the DNA High-Sensitivity LabChip assay (Agilent Technologies, Santa Clara, CA) and quantitative PCR (KAPA Biosystems, Wilmington, MA), according to the manufacturers' instructions. RIP-seq libraries were pooled and sequenced on a MiSeq 50 base pair single-read flow cell using v2 reagents (Illumina, San Diego, CA). SIR libraries were also pooled and sequenced on either a HiSeq2500 high output 100 base pair paired-end flow cell using TruSeq SBS v3 reagents or on a NextSeq500 high output 75 base pair single-read flow cell using v2 reagents (Illumina, San Diego, CA).