Materials and Reagents

Whatman filter paper (Merk, catalog number: WHA1001150)
Pipette tip 1,000 μl (Axygen, catalog number: T-1000-C-L-R-S)
Pipette tip 10 ml (Eppendorf, catalog number: 0030000781)
1.5 ml microcentrifuge tube (Axygen, catalog number: MCT-150-C-ZX)
50 ml centrifuge tube (Corning, catalog number: 430828)
Petri dish (any brand, 47 mm diameter)
Funnel (any brand, 100 mm diameter)
Arabidopsis ddm1-2 mutant in the Columbia-0 background
Ethanol (Merk, catalog number: 51976)
Triton X-100 (Merk, catalog number: T8787)
Murashige and Skoog basal medium with Vitamins (PhytoTech, catalog number: M519)
Distilled water, generated using the RSJ Water Purification system (Tanon, catalog number: RODI-220B1)
Liquid nitrogen
Miracloth (Sigma-Aldrich, catalog number: 475855)
Tris base (Fisher Scientific, catalog number: BP152-500)
Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473)
Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A466-250)
Potassium acetate (KOAc) (Sigma-Aldrich, catalog number: P1190)
Magnesium acetate (MgOAc) (Sigma-Aldrich, catalog number: 63052)
Dithiothreitol (DTT) (Fisher Scientific, catalog number: R0861)
Nonylphenyl-polyethylene Glycol (NP-40) (Fisher Scientific, catalog number: 49-201)
cOmplete^TM, EDTA-free protease inhibitor cocktail (Sigma-Aldrich, catalog number: 11873580001)
RNasin^® Plus RNase inhibitor (Promega, catalog number: N2615)
Sterilization solution (see Recipes)
Half-strength MS-medium plate (see Recipes)
RG lysis buffer (see Recipes)

Equipment

Pipette 1,000 μl, 10 ml (Eppendorf, catalog numbers: 3123000063, 4720000011)
Vortexer (any brand)
Clean bench (any brand)
Refrigerator (4°C) (any brand)
Plant growth chamber (any model with temperature and light control)
Mortar and pestle (any brand)
Sorvall LYNX 4000 Superspeed Centrifuge (ThermoFisher, catalog number: 75006580)
Microcentrifuge 5424R (Eppendorf, model: 5424R)
Centrifuge 5810R (Eppendorf, model: 5810R, catalog number: 022625101)

Procedure

Overview
This protocol allows for the simple and fast enrichment of RG fractions (albeit crude) from plant samples. Briefly, the RG fractions are separated and dissolved by continuous centrifugation with RG lysis buffer. The enriched RG fractions can be subsequently subjected to RNA-seq and protein analysis (Figure 1).

Figure 1. Scheme of RNA granule enrichment

Plant preparation
1. Place 100 μl of ddm1-2 seeds in a 1.5 ml microcentrifuge tube.
2. Add 1 ml of sterilization solution to seeds and vortex for 4 min (see Recipe 1).
3. Discard the solution.
4. Wash the seed with 1 ml of 100% ethanol.
5. Vortex for 1 min and discard the ethanol.
6. Repeat Steps B4 and B5.
7. Prepare a sheet of filter paper and pipet the seeds onto the paper.
8. Let the ethanol evaporate for 3 min.
9. Pick up the paper and sprinkle the seeds onto half-strength Murashige and Skoog medium (see Recipe 2).
10. Wrap the plate with parafilm and place it into a 4°C chamber for 2 days.
11. Transfer the plate into a growth chamber set at 22°C and under 16 h light/8 h dark cycles for 10 days.
Notes:
1. Any plant samples in addition to Arabidopsis seedlings can be used for this protocol.
2. Perform seed sterilization on a clean bench (wipe down with 70% ethanol before use).

RNA granule enrichment
1. Grind 2 g of seedlings into a fine powder in liquid nitrogen using a precooled mortar and pestle.
2. Collect the samples (approximately 5 ml) into a 50 ml tube and resuspend in 5 ml of RG lysis buffer (see Recipe 3).
3. Filter the resulting slurry through four layers of Miracloth in a funnel to a 50 ml conical tube and centrifuge at 850 × g for 5 min at 4°C to pellet cell debris.
4. Transfer the supernatant to a new 50 ml tube and add 5 ml of RG lysis buffer.
5. Centrifuge at 4,000 × g for 10 min at 4°C and discard the supernatant.
6. Resuspend the pellet in 2 ml of RG lysis buffer. Centrifuge at 18,000 × g for 10 min at 4°C.
7. Resuspend the pellet in 2 ml of RG lysis buffer, vortex, and centrifuge at 18,000 × g at 4°C for 10 min.
8. Discard the supernatant and resuspend the pellets gently in 1 ml of RG lysis buffer. Centrifuge at 850 × g for 10 min at 4°C.
9. Transfer the supernatant (enriched with RGs) into a 1.5 ml microcentrifuge tube without disturbing any residue and keep it in a freezer until use.
Note: We recommend using the fluorescence-tagged RG-marker plant lines to quickly check for successful RG enrichment.

RNA analysis
The final RG fraction resulting from the protocol described above can be subjected to regular RNA extraction and subsequently tested for either targeted RNA analyses using RT-qPCR or transcriptome-wide profiling with RNA-seq.
Note: Refer to Kim et al. (2021) for suggestions on any RG-specific marker genes.

Data analysis

RNA-seq data generated from the RG enrichment fractions can be analyzed as detailed in the original paper (Kim et al., 2021; https://doi.org/10.1038/s41477-021-00867-4).

Recipes

Sterilization solution
70% ethanol
0.05% Triton X-100
Half-strength MS-medium plate
2.2 g/L Murashige and Skoog basal medium with Vitamins
Adjust pH to 5.7 with KOH
7 g/L plant agar
Sterilize by autoclaving
RG lysis buffer
50 mM Tris-HCl, pH 7.4
100 mM KOAc
2 mM MgOAc
0.5% NP-40
0.5 mM DTT
One tablet (in 50 ml) of protease inhibitor cocktail (cOmplete^TM, EDTA-free Protease Inhibitor Cocktail)
1 U/μl RNasin Plus RNase Inhibitor

1 M Tris-HCl, pH 7.4 2.5 ml
1 M KOAc 5 ml
1 M MgOAc 0.1 ml
10% NP-40 2.5 ml
1 M DTT 25 μl
Protease inhibitor cocktail One tablet
40,000 U/ml RNasin Plus RNase Inhibitor 1.25 ml
Distilled water Top up to 50 ml

Notes:

Prepare all solutions and buffers with distilled water.
Add DTT and RNase Inhibitor right before use.

Acknowledgments

The work was supported by grants from the National Natural Science Foundation of China (31970518), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB27030209), and the General Program of Natural Science Foundation of Shanghai (21ZR1470700). E. Y. Kim is the recipient of a President’s International Fellowship Initiative (PIFI) young staff fellowship (2021FYB0001) from CAS. This protocol was adapted from our previously reported work (Kim et al., 2021).

Competing interests

The authors declare no conflicts of interest.

References

Anderson, P. and Kedersha, N. (2009). RNA granules: post-transcriptional and epigenetic modulators of gene expression. Nat Rev Mol Cell Biol 10(6): 430-436.
Jain, S., Wheeler, J. R., Walters, R. W., Agrawal, A., Barsic, A. and Parker, R. (2016). ATPase-modulated stress granules contain a diverse proteome and substructure. Cell 164(3): 487-498.
Khong, A., Matheny, T., Jain, S., Mitchell, S. F., Wheeler, J. R. and Parker, R. (2017). The stress granule transcriptome reveals principles of mRNA accumulation in stress granules. Mol Cell 68(4): 808-820e805.
Kim, E. Y., Wang, L., Lei, Z., Li, H., Fan, W. and Cho, J. (2021). Ribosome stalling and SGS3 phase separation prime the epigenetic silencing of transposons. Nat Plants 7(3): 303-309.
McCue, A. D., Nuthikattu, S., Reeder, S. H. and Slotkin, R. K. (2012). Gene expression and stress response mediated by the epigenetic regulation of a transposable element small RNA. PLoS Genet 8(2): e1002474.
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