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Immunoprecipitation of piRNPs and Directional, Next Generation Sequencing of piRNAs

  • Yohei Kirino
  • Anastassios Vourekas
  • Eugene Khandros
  • Zissimos Mourelatos
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 725)

Abstract

Piwi interacting RNAs (piRNAs) are small (∼25 to ∼30 nucleotide) and are expressed in the germline. piRNAs bind to the Piwi subclade of Argonaute proteins and form the core ribonucleoproteins (RNPs) of piRNPs. We describe a method for the massive identification of piRNAs from immunopurified piRNPs. This strategy may also be used for immunopurification and directional sequencing of RNAs from other RNPs that contain small RNAs.

Key words

piRNA piRNP Argonaute Piwi Xili Xiwi Mili Miwi Miwi2 Aub Ago3 Y12 Next gen sequencing Illumina cDNA Immunoprecipitation RNA-Immunoprecipitation RNA-IP T4 RNA ligase Reverse Transcriptase Polymerase Chain reaction PCR RT-PCR Posttranscriptional RNA processing Gene silencing 

Notes

Acknowledgment

Supported by NIH grants GM072777 and NS056070 to ZM.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Yohei Kirino
  • Anastassios Vourekas
  • Eugene Khandros
  • Zissimos Mourelatos
    • 1
  1. 1.Division of Neuropathology, Department of Pathology and Laboratory MedicineUniversity of Pennsylvania School of MedicinePhiladelphiaUSA

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