TITLE: PHYTOMap in Arabidopsis root tips
AUTHORS: Tatsuya Nobori, Joseph Ecker

[DESCRIPTION]
Retrieving the complex responses of individual cells in the native three-dimensional tissue context is crucial for a complete understanding of tissue functions. Here, we present PHYTOMap (Plant HYbridization-based Targeted Observation of gene expression Map), a multiplexed fluorescencein situhybridization method that enables single-cell and spatial analysis of gene expression in whole-mount plant tissue in a transgene-free manner and at low cost. We applied PHYTOMap to simultaneously analyze 28 cell type marker genes inArabidopsisroots and successfully identified major cell types, demonstrating that our method can substantially accelerate the spatial mapping of marker genes defined in single-cell RNA-seq datasets in complex plant tissue.

 

[Reagents]

DPBST
0.1% (vol/vol) Tween-20 in 1x DPBS

DPBSTR
DPBST + 1:100 SUPERaseIN

Cell wall digestion enzyme solution (CWDES; 10x stock)
250 mg macerozyme, 250 mg cellulase, 500 mg pectinase in 50 mL Nuclease-free water. Filter sterilize (0.22 μm filter) and store aliquots of 1 mL at -20°C.

Proteinase K buffer
ABReagentAmount10x equiPhi29 DNA polymerase buffer10 µL10 mM dNTP2.5 µL4 mM aminoallyl-dUTP0.5 µLBSA (20 mg/ml)0.5 µLDTT (100 mM)1 µLSUPERase IN1 µLNuclease Free Water 84.5 µL
 
FAA
ABReagentAmount1M Tris-HCl (pH 8.0)5 mL0.5 M EDTA (pH 8.0)5 mLNuclease Free Water up to 50 mL
 

 


[GUIDELINES]
See the following paper for more information on PHYTOMap. 

PHYTOMap: Multiplexed single-cell 3D spatial gene expression analysis in plant tissue
Tatsuya Nobori, Marina Oliva, Ryan Lister, and Joseph R. Ecker, bioRxiv, 2022
DOI: 10.1101/2022.07.28.501915

To avoid RNase contamination, special precautions are necessary when handling samples in this protocol. It is advisable to allocate a dedicated area and equipment specifically for RNA work, and to clean them with commercial RNase and DNAse inactivating agents before wiping them with ethanol.
Probes have to be accurately designed. Detailed information on probe design is available in the publication above.



[STEPS]
SECTION: Sampling
1. Cut Arabidopsis root tips (approximately 1 cm) with razor blade and place them on a Poly-D-Lysine coated dish. The tissue should adhere to the dish well.
SECTION: Tissue Permeabilization
5. 
Rehydrate the tissue by 5 min successive washes with 100 µL 75%, 50%, 25% MeOH in DPBST. 
SECTION: Gene Specific Probe Hybridization
15. Mix gene specific probes at the concentration of 5 nM per oligo. 
SECTION: Rolling Circle Amplification  (RCA)
23. Wash the tissue with 100 µL DPBSTR for 5 min.

SECTION: Target Detection with Sequence-By-Hybridization
34. Wash the tissue twice with100 µL 2xSSC for 1 min.

SECTION: Tissue Fixation
2. Incubate the tissue in 100 µL of FAA (see MATERIALS) at Room temperature for 60 min. 

 

SECTION: Tissue Fixation
3. Wash the tissue successively for 10 min at Room temperature with 100 µL 70% EtOH, 90% EtOH, and twice with 100% EtOH.

SECTION: Tissue Fixation
4. Wash the tissue twice with 100 µL 100% MeOH for 10 min and leave the tissue in MeOH after the second wash at -20 °C 30 min. 

SECTION: Tissue Permeabilization
7. Remove the CWDES and add 100 µL fresh & cold 1x CWDES. Then, incubate the tissue at Room temperature for 30 min.
SECTION: Tissue Permeabilization
8. Wash the tissue twice with 100 µL DPBSTR (see MATERIALS).

SECTION: Tissue Permeabilization
9. Fix the tissue by incubating in 100 µL 10% (v/v) formaldehyde in DPBST at Room temperature for 30 min.

SECTION: Tissue Permeabilization
10. Wash the tissue twice with 100 µL DPBSTR at Room temperature (1 min each).

SECTION: Tissue Permeabilization
11. Incubate the tissue in 100 µL digestion solution at 37 °C for 30 min.

 

SECTION: Tissue Permeabilization
12. Wash the tissue twice with 100 µL DPBSTR at Room temperature (1 min each).

SECTION: Tissue Permeabilization
13. Fix the tissue by incubating in 100 µL 10% (v/v) formaldehyde in DPBST at Room temperature for 30 min.

SECTION: Tissue Permeabilization
14. Wash the tissue twice with 100 µL DPBSTR at Room temperature (5 min each).

SECTION: Gene Specific Probe Hybridization
16. Heat the probe mixture at 90 °C for 3 min and let it cool down at Room temperature. 

SECTION: Gene Specific Probe Hybridization
17. Incubate the tissue in the hybridization mixture at 40 °C for 180 min or 180 min

 
ABReagentAmount20xSSC10 µLFormamide30 µL10% Triton-X10 µL200 mM RVC10 µLSUPERase IN1 µLProbe mix (500 nM per oligo)2 µLNuclease Free Water 37 µL
 

 

SECTION: Gene Specific Probe Hybridization
18. Wash the tissue twice with 100 µL DPBSTR at 37 °C for 30 min.

SECTION: Gene Specific Probe Hybridization
19. Wash the tissue twice with 100 µL 4xSSC in DPBSTR at 37 °C for 30 min.

 
ABReagentAmount20xSSC10 µLFormamide30 µL10% Triton-X10 µL200 mM RVC10 µLSUPERase IN1 µLProbe mix (500 nM per oligo)2 µLNuclease Free Water 37 µL
 

SECTION: Gene Specific Probe Hybridization
20. Rinse the tissue with 100 µL DPBSTR at Room temperature.

SECTION: Ligation
21. Incubate the tissue in 100 µL ligation mixture WITHOUT ligase on ice for 5 min.

 

SECTION: Ligation
22. Incubate the tissue in 100 µL ligation mixture WITH ligase at Room temperature  .
 
ABReagentAmount10x ligation buffer10 µLBSA (2mg/ml)0.5 µLSUPERase IN1 µLNuclease Free Water 88.5 µL
 
SECTION: Rolling Circle Amplification  (RCA)
24.  Incubate the tissue in 100 µL RCA mixture WITHOUT equiPhi29 DNA polymerase on ice for 5 min.

ABReagentAmount10x ligation buffer10 µLBSA (2mg/ml)0.5 µLSUPERase IN1 µLNuclease Free Water 88.5 µL
 



SECTION: Rolling Circle Amplification  (RCA)
25. Incubate the tissue in 100 µL RCA mixture WITH equiPhi29 DNA polymerase at 37 °C 5 min. 

ABReagentAmount10x equiPhi29 DNA polymerase buffer10 µL10 mM dNTP2.5 µL4 mM aminoallyl-dUTP0.5 µLBSA (20 mg/ml)0.5 µLDTT (100 mM)1 µLSUPERase IN1 µLNuclease Free Water 84.5 µL

SECTION: Rolling Circle Amplification  (RCA)
26. Wash the tissue twice with 100 µL DPBSTR for 10 min.

SECTION: Post-Amplification Fixation
27. Incubate the tissue in 100 µL BS(PEG9) solution at Room temperature for 60 min.
 
ABReagentAmount10x equiPhi29 DNA polymerase buffer10 µL10 mM dNTP2.5 µL4 mM aminoallyl-dUTP0.5 µLBSA (20 mg/ml)0.5 µLDTT (100 mM)1 µLSUPERase IN1 µLequiPhi29 DNA polymerase5 µLNuclease Free Water 79.5 µL
 

 

SECTION: Post-Amplification Fixation
28. Aspire BS(PEG9) solution and incubate the tissue in 100 µL 1 M Tris-HCl pH 8.0 at Room temperature for 30 min.

SECTION: Post-Amplification Fixation
29. Rinse the tissue in 100 µL DPBST.


 


SECTION: Gel Embedding
30. Incubate the tissue in 100 µL monomer solution on ice for 30 min.
 
ABReagentAmountDPBST196 µLBS(PEG9) stock4 µL
 
SECTION: Gel Embedding
31. Aspire the monomer solution and add 50 µL gelling solution. Place a Gel Slick-coated glass coverslip on top of the tissue, and carefully aspirate any excess gelling solution. Incubate the tissue at Room temperature for 60 min––120 min until the gel solidifies.
 
ABReagentAmount20% acrylamide20 µL2% bis-acrylamide10 µL2xSSC70 µL
 

 

SECTION: Gel Embedding
32. Carefully remove the coverslip with forceps, and wash the tissue with 100 µL DPBST for 5 min.

SECTION: Gel Embedding
33. Incubate the tissue in ClearSee until imaging. 
SECTION: Target Detection with Sequence-By-Hybridization
35. Incubate the tissue in 100 µL Bridge probe mixture at Room temperature for 60 min.
  
ABReagentAmountMonomer solution48.9 µL10% APS stock1 µLTEMED0.1 µL
 

SECTION: Target Detection with Sequence-By-Hybridization
36. Wash the tissue twice with 100 µL 2xSSC for 1 min.

SECTION: Target Detection with Sequence-By-Hybridization
37. Incubate the tissue in 100 µL Detection probe mixture at Room temperature for 60 min.
 
ABReagentAmount2x hybridization buffer50 µLBridge probes (10uM stock) 1 µL each (typically 4 probes)Nuclease free water up to 100 µL
 
SECTION: Target Detection with Sequence-By-Hybridization
38. Wash the tissue twice with 100 µL 2xSSC for 1 min.

SECTION: Target Detection with Sequence-By-Hybridization
39. Wash the tissue with 100 µL ClearSee, and keep it in ClearSee for imaging.

SECTION: Target Detection with Sequence-By-Hybridization
40. Imaging with a confocal microscope.
SECTION: Target Detection with Sequence-By-Hybridization
41. After the imaging, strip the bridge/detection probes by incubating the tissue in100 µL stripping solution (65% formamide in 2xSSC) at 30 °C for 30 min.
 
ABReagentAmount2x hybridization buffer50 µLDetection probes (10uM stock) 1 µL each (4 probes)Calcofluor White1 µLNuclease free water up to 100 µL
 
42. Go to Step 35 for the next round of imaging.
SECTION: Tissue Permeabilization
6. Incubate the tissues in 100 µL 1x CWDES (see MATERIALS) on ice for 5 min.

 
ABReagentAmount10x CWDES10 µLSUPERase IN1 µLDPBST89 µL