Background

MicroRNAs (MiRNAs) play an important role in many cellular growth and development processes. A fantastic feature of miRNAs is the non-cell autonomous action, in which miRNA moves from cell to cell or over long distances (Melnyk et al., 2011), serving as a signal molecule that functions locally or systemically. Previous studies have shown that the long distance or systemic movement of miRNA is mainly through the phloem, while cell-to-cell movement is through plasmodesmata (Melnyk et al., 2011). Mobile miRNAs, such as miR394 and miR165/6, have been shown to serve as morphogens, which shape the cell pattern formation during development (Breakfield et al., 2012;  Carlsbecker et al., 2010;  Knauer et al., 2013). Root cell layer-specific promoter-driven expression of AGO1, the core protein in the miRNA-induced silencing complex (miRISC), shows that AGO1 is cell autonomous (Brosnan et al., 2019;  Fan et al., 2022). Microtubules have been shown to specifically regulate the association of miRNA165/6 with AGO1 in the cytoplasm and play a vital role in miRNA cell-to-cell movement (Fan et al., 2022). Therefore, characterization of miRNA distribution at a high spatial resolution is critical for understanding their functions in gene regulation. Here, we describe a protocol to profile cell-type-specific miRNA by immunoprecipitation of root cell layer-specific green fluorescent protein (GFP)-AGO1, followed by small RNAseq, that provides a new tool to deepen our understanding of the regulation of miRNA functions.

Materials and Reagents

1. RNase-free pipette tips [Genesee Scientific, catalog numbers: 23-130R (10 µL), 23-150R (200 µL), 23-165R (1,250 µL)]

2. 1.5 mL centrifuge tubes (Genesee Scientific, catalog number: 22-282)

3. 50 mL centrifuge tubes (Genesee Scientific, catalog number: 21-108)

4. 15 mL centrifuge tubes (Genesee Scientific, catalog number: 21-103)

5. Petri dishes (Genesee Scientific, catalog number: 32-106)

6. Nylon mesh (mesh opening sizes 100 µm) (NITEX, catalog number: NTX100-098)

7. Liquid nitrogen

8. Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, catalog number: F-530XL)

9. In-Fusion HD cloning plus (Takara, catalog number: 638911)

10. Murashige and Skoog medium (MS) (PhytoTech Lab, catalog number: M524)

11. IGEPAL CA-630 (MilliporeSigma, catalog number: I8896)

12. GFP-Trap magnetic agarose (ChromoTek, gtd-20)

13. Sodium dodecyl sulfate (SDS) (MilliporeSigma, catalog number: L3771-500G)

14. β-mercaptoethanol (MilliporeSigma, M3148-250ML)

15. Glycerol (Thermo Fisher Scientific, catalog number: J16374.K2)

16. Glycogen RNA grade (Thermo Fisher Scientific, catalog number: R0551)

17. NEBNext multiplex small RNA library prep set for Illumina (New England Biolabs, catalog number: E7300)

18. 10 bp O'RangeRuler DNA ladder (Thermo Fisher Scientific, catalog number: SM1313)

19. Sodium chloride (NaCl) (Thermo Fisher Scientific, catalog number: AAJ2161836)

20. DEPC-treated water (Thermo Fisher Scientific, catalog number: 4387937)

21. Trizma base (MilliporeSigma, catalog number: T1503)

22. TRI reagent (MRC, catalog number: TR118)

23. Clorox germicidal bleach (Thermo Fisher Scientific, catalog number: Essendant CLO30966EA)

24. Triton X-100 solution (MilliporeSigma, catalog number: 93443)

25. Bromophenol blue (MilliporeSigma, catalog number: B0126)

26. cOmplete, EDTA-free protease inhibitor cocktail (MilliporeSigma, catalog number: 11836170001)

27. GFP antibody (MilliporeSigma, catalog number: 11814460001)

28. Chloroform (Thermo Fisher Scientific, catalog number: 423550040)

29. Isopropanol (Thermo Fisher Scientific, catalog number: 423835000)

30. 6× DNA loading dye (Thermo Fisher Scientific, catalog number: R0611)

31. Ethidium bromide (Thermo Fisher Scientific, catalog number: J67270.14)

32. Sodium acetate (Thermo Fisher Scientific, catalog number: A13184.30)

33. Seed sterilization solution (see Recipes)

34. 2× protein SDS loading buffer (see Recipes)

35. IP lysis buffer (see Recipes)

36. Washing buffer (see Recipes)

37. 5× TBE buffer (see Recipes)

38. 12% polyacrylamide gel (see Recipes)

39. 8% SDS-PAGE resolving gel (see Recipes)

40. SDS-PAGE stacking gel (see Recipes)

41. 3 M sodium acetate (pH 5.2) (see Recipes)

Equipment

1. Plant growth chamber (Percival, model: CU36L5)

2. Microcentrifuge (Eppendorf, model: 5424R)

3. High-speed centrifuge (Beckman Counter, model: Avanti J-E Series)

4. DynaMag-2 magnet (Invitrogen, catalog number: 12321D)

5. H5600 revolver tube mixer (VWR, catalog number: H5600-15)

6. Thermocyclers (Bio-Rad, catalog number: 1851148)

7. Blade

8. Thermomixer C (Eppendorf, catalog number: 5382000015)

9. Spatula

10. Mortar and pestle

11. Mini-PROTEAN tetra vertical electrophoresis cell (Bio-Rad, catalog number: 1658001FC)

Procedure