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RNA Immunoprecipitation (RIP) Sequencing of Pri-miRNAs Associated with the Dicing Complex in Arabidopsis

ZL Ziwei Li
SZ Songxiao Zhong
WW Wenye Wu
BZ Binglian Zheng

Published: Jul 5, 2018 DOI: 10.21769/BioProtoc.2909 Views: 6917

Edited by: Amey Redkar Reviewed by: Marisa Rosa

Original research article

The authors used this protocol in:

http://www.ncbi.nlm.nih.gov/pubmed/27870853

Nov 2016

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Abstract

RNA immunoprecipitation (RIP) is an antibody-based technique used to map in vivo RNA-protein interactions. DBR1, an RNA debranching enzyme, is responsible for the debranching of lariat RNA, for the degradation and turnover of lariat RNAs. It is well known that primary miRNA (Pri-miRNA) is recognized and further processed into mature miRNA by the Dicing complex mainly composed of DCL1 and HYL1. Due to the low abundance of pri-miRNAs, RIP followed qRT-PCR has been widely used to evaluate the binding efficiency of the Dicing complex with pri-miRNAs in previous studies. Therefore, the genome-wide evaluation of the Dicing complex with pri-miRNAs is lacking. With the improvement of high-throughput sequencing technologies, we successfully used RIP-seq to compare the binding efficiency of the Dicing complex with pri-miRNAs between wild-type and the dbr1-2 mutant in our recent study. In this protocol, we provide a detailed description of RIP-seq using GFP-trap beads in HYL1-YFP and DCL1-YFP transgenic plants between two different genotypes. This method can be used to assess the binding of pri-miRNAs with the Dicing complex in Arabidopsis, and it can be applied to other RNA binding proteins in plants.

Keywords: DBR1 search RIP search Pri-miRNA search The Dicing complex search

Materials and Reagents

  1. RNase-free pipette tips
  2. RNase-free Eppendorf tubes (Safe-Lock Tubes, Eppendorf, catalog number: 0030120086 )
  3. 50 ml and 15 ml RNase-free Falcon tubes (Corning Company)
  4. Miracloth (Merck, catalog number: 475855-1R )
  5. Paper towels
  6. Fresh inflorescences including all buds and open flowers together (see Figure 1) of proHYL1::YFP-HYL1/Col-0, proHYL1::YFP-HYL1/dbr1-2, proDCL1::DCL1-YFP/Col-0, proDCL1::DCL1-YFP/dbr1-2 plants grown in 16 h/8 h light/dark condition, 22 °C


    Figure 1. A mature plant with the position of the tissue used in this method indicated by a red rectangle. Inflorescences were collected freshly. In general, it is better to use the inflorescences from primary shoot for this experiment.

  7. Liquid nitrogen
  8. Ice
  9. 37% formaldehyde (Sinopharm Chemical Regent, catalog number: 10010018 , store in the dark)
  10. Diethy pyrocarbonate (DEPC) (Sigma-Aldrich, catalog number: D5758 )
  11. GFP-Trap agarose beads (Chromotek, catalog number: gta-20 )
  12. Protein A agarose/Salmon Sperm DNA (Merck, Upstate, catalog number: 16-157 )
  13. RNase inhibitor (Promega, catalog number: N2611 )
  14. RQ1 DNase I (Promega, catalog number: M6101 )
  15. Glycogen (Thermo Fisher Scientific, catalog number: R0551 )
  16. Proteinase K (Thermo Fisher Scientific, catalog number: EO0491 )
  17. SuperScript IV (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18090200 )
  18. Taq DNA polymerase (Takara Bio, catalog number: R500Z )
  19. dNTP (Thermo Fisher Scientific, catalog number: R0181 )
  20. Oligo dT (Thermo Fisher Scientific, catalog number: SO132 )
  21. DTT (Thermo Fisher Scientific, catalog number: R0861 )
  22. Acidic Phenol:Chloroform
    1. Chloroform (Acros Organics, catalog number: 423555000 )
    2. Acidic Phenol (Sangon Biotech, catalog number: A504195 )
  23. Ethanol (Acros Organics, catalog number: 397690010 )
  24. Agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500100 )
  25. PMSF (Sigma-Aldrich, catalog number: P7626 )
  26. Protease Inhibitor Cocktail (Roche Diagnostics, catalog number: 11873580001 )
  27. Illumina TruSeq Stranded Total RNA HT Sample Prep Kit (P/N15031048)
  28. MgCl2 (Sangon Biotech, catalog number: MB0331 )
  29. CaCl2 (Sangon Biotech, catalog number: CT1330 )
  30. Glycine (Sangon Biotech, catalog number: A610235 )
  31. EDTA (Sangon Biotech, catalog number: A100105 )
  32. NaAc (Sangon Biotech, catalog number: ST0827 )
  33. Sucrose (Sangon Biotech, catalog number: SB0498 )
  34. HEPES (Sangon Biotech, catalog number: H0511 )
  35. Tris (Sangon Biotech, catalog number: A600194 )
  36. SDS (Sigma-Aldrich, catalog number: L5750 )
  37. NaCl (Sangon Biotech, catalog number: A501218 )
  38. Ficoll (Sigma-Aldrich, catalog number: F2637 )
  39. Dextran T40 (Sangon Biotech, catalog number: DB0374 )
  40. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  41. Primers used in Figure 3:
    Pri-miR156a-F: CAAGAGAAACGCAAAGAAACTGACAG
    Pri-miR156a-R: AAAGAGATCAGCACCGGAATCTGACAG
    Pri-miR158a-F: GTGATGACGCCATTGCTCTTT
    Pri-miR158a-R: TGTGACTTTAGATGCCCTTGTTCA
    Pri-miR159a-F: GGAGCTCTACTTCCATCGTCA
    Pri-miR159a-R: CCACGTTCTCATCAAAACTTTC
    Pri-miR166a-F: GACTCTGGCTCGCTCTATTCA
    Pri-miR166a-R: TGGTCCGAAGACGCTAAAAC
    Pri-miR167a-F: GAAGCTGCCAGCATGATCTA
    Pri-miR167a-R: GGGTTTATAGAAGGGTGCGA
    Pri-miR168aF: GCCTTGCATCAACTGAAT
    Pri-miR168aR: CAAACAAAAGGAGACTAAAGA
    Pri-miR169aF: TGGGTATAGCTAGTGAAACGCG
    Pri-miR169aR: CCTTAGCTTGAGTTCTTGCGA
    Pri-miR171aF: CCGCGCCAATATCTCAGTA
    Pri-miR171aR: TGTCTCCATTTCAACACACACA
    Pri-miR172aF: ATCTGTTGATGGACGGTGGT
    Pri-miR172aR: AATAGTCGTTGATTGCCGATG
    Pri-miR319aF: GAGATAGAGAGTTGAACAAATTCTTC
    Pri-miR319aR: GTATCCATGATAGTTGAGAAATTTGC
  42. Honda Buffer (see Recipes)
  43. Nuclei Lysis Buffer(see Recipes)
  44. ChIP Dilution Buffer (see Recipes)
  45. Binding/Washing Buffer (see Recipes)
  46. RIP Elution Buffer (see Recipes)

Equipment

  1. Pipettes (Gilson Company)
  2. Fume hood
  3. Vacuum Pump (Shanghai SENCO Company, model: SHB-3 )
  4. Microcentrifuge (Thermo)
  5. Vortexer (VWR Company)
  6. Mortar and Pestle (Fisher)
  7. -80 °C Freezer (Thermo)
  8. Rotator (Haimen Kylin-Bell Lab Instruments, catalog number: KB-3-D )
  9. Mini Gel Electrophoresis Systems (Tanon Company)
  10. ChIP-grade Sonifier (Diagenode Bioruptor)
  11. Illumina HiSeq 2000 (Shanghai Genegy Company)

Procedure

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.

Category

Plant Science > Plant molecular biology > RNA

Molecular Biology > RNA > RNA-protein interaction

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