GSM7818300: IFIH1-RIP; Mus musculus; RNA-Seq
RNA extraction and sequencing: Total RNA was extracted from the isolated BMDMs using TRIzol. After rRNA depletion, the remaining RNA was prepared for library preparation following Illumina's instructions. The resulting libraries were sequenced using Illumina Sequencing Technology. RIP-seq for IFIH1-bound RNA complexes: RNA extracts from the isolated BMDMs were subjected to RNA-binding protein immunoprecipitation (RIP) using the Magna RIPTM kit. The IFIH1-bound RNA complexes were enriched by incubating the RNA extracts with IFIH1 antibody-bound beads and RIP immunoprecipitation buffer. The purified RNAs from the IFIH1-bound complexes were then sequenced using Illumina Sequencing Technology, with 5% input RNA as a negative control to reduce noise. Provide at least 1 ug (2-5 ug preferred) of total RNA at a concentration of at least 50 ng/ul (1 ug for Poly-A enrichment; 2 ug for ribo-depletion libraries; using less starting material is possible, but we can't guarantee results). Please make sure that your RNA isolation protocol employs a DNAse digestion step or other means to remove DNA from the sample. On an agarose gel, DNA contamination will be visible as a smear of band of fragments considerably larger than the RNA (>10 kb).  To verify the purity of the RNA samples the 260/280 ratio should be between 1.8 and 2.1 and 260/230 ratio should be higher than 1.5.  Poly-A enrichment, ribo-depletion and strand specific library prep are among the commonly requested types of service (more technical details on this appear below). Libraries for slightly degraded RNA samples should be prepared using ribo-depletion protocols. If possible please avoid RNA extraction protocols involving Trizol or related phenol containing reagents (silica column based kits are less likely to retain contaminants). If using Trizol, protocols that contain a column based cleanup (e.g. Direct-zol, TRIzolPlus) have to be used.  Please note that an additional column cleanup is mandatory for RNA samples isolated from PAXgene tubes or with PAXgene kits. RNA samples should be eluted in molecular biology grade water, always stored in a -80 degree freezer and shipped on dry ice.  All RNA samples require a Bioanalyzer sample QC (or equivalent).  Such QC traces can be submitted by the customers or we can run the QC  for a fee instead.