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RIP-Seq

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The Introduction of RIP-Seq

RIP-Seq

Every activity of transcript lifecycle, from birth (polymerases) to degradation (nucleases), involves protein binding. In addition to protein production, a subset of these transcripts plays crucial roles in other essential processes such as epigenetic regulation and genome protection by transposon silencing. Most studies have focused on transcriptomics profiling. It is believed, however, that the levels of mRNAs do not always directly correlate with the steady-state protein levels. Interest in identifying the RNAs associated with RNA binding protein (RBP) in a cellular context is growing as the role of RNA processing and translational events that occur post-transcriptionally is begin to be appreciated.

RIP-Seq maps the sites at which proteins are bound to the RNA within RNA-protein complexes. RNA immunoprecipitation (RIP) implies the purification of RNA–protein interactions in native conditions by using a protein-specific antibody to map the RBP of interest. The advent of sequencing technologies, coupled to various RIP chemistries, has enabled the simultaneous detection of thousands bound transcripts (mRNAs, non-coding RNAs or viral RNAs) in a single experiment. CD genomics provides RIPed RNA sequencing to obtain insights into not just the well-established processes such as transcription, splicing and translation, but also in newer fields such as RNA interference and gene regulation by non-coding RNAs.

RIP-Seq workflow

CD Genomics applies the illumina NGS equipment to sequence the bound transcripts, unveiling genome-wide interactions of RBPs. Researchers will submit the RNA pulled by the appropriate RIP methods based on specific protein immunoprecipitation from cell lines or tissues. Our RIP-Seq service, offering the workflow from sample QC through data analysis, enables rapid profiling and deep insight of the RNA.

RIP-Seq WorkflowFig2. RIP-Seq Workflow

Service Specifications

Sample requirements

  • Fragment sizes: 100 – 800 bp
  • Immunoprecipitated RNA > 50 ng, concentration > 10 ng/ul, OD260/280 1.8-2.0
Sequencing

Flexible service options include HiSeq 4000HiSeq X ten and NextSeq 500

Bioinformatics Analysis

  • Raw data QC
  • Alignment and TPM/RPKM/FPKM-based quantitation
  • Expression analysis
  • Alternative splicing analysis
  • GO and KEGG annotation

Deliverables

  • The original sequencing data
  • Experimental results
  • Data analysis report
  • Details in Single-Cell Sequencing for your writing (customization)

If you have additional requirements or questions, please feel free to contact us.

Reference
Zhao J et al. Genome-wide identification of polycomb-associated RNAs by RIP-seq. Molecular Cell, 2010 Dec 22;40(6):939-53

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