GSM5888885: SKNAS WT KIF5A RIP1; Homo sapiens; RIP-Seq
RNA was extracted from immunoprecipitation elution samples using Trizol reagent. For the IP, conjugated beads were used, the Anti-V5 Tagged Magnetic Beads (MBL International Corp, Cat# M167-11). RNA transcripts are enriched by selectively depleting ribosomal RNA molecules using the Ribogone Low-input-Mammalian kit (Clontech Cat. Nos. 634846 & 634847 ), which allows for the specific removal of rRNA sequences (5S, 5.8S, 18S, and 28S), as well as mitochondrial rRNA sequences (12S), from human, mouse, or rat total RNA. Fragmentation times are adjusted based on the RIN value for each sample so that an insert size of 150-200 bp can be achieved.  The SMARTer Stranded RNA-Seq Kit (Cat. Nos. 634836) is then used to generate a cDNA library.  The cDNA library is then amplified and indexed adapters are added.  Libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the Perkin Elmer LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/ul are used for sequencing.