PHYTOMap in Arabidopsis root tips
Joseph Ecker, Tatsuya Nobori
Abstract
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 fluorescence in situ hybridization 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 in Arabidopsis roots 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
| A | B |
|---|---|
| Reagent | Amount |
| 1M Tris-HCl (pH 8.0) | 5 mL |
| 0.5 M EDTA (pH 8.0) | 5 mL |
| Nuclease Free Water | up to 50 mL |
FAA
| A | B |
|---|---|
| Reagent | Amount |
| 32% formaldehyde | 450 µL |
| Acetic acid | 50 µL |
| Ethanol | 500 µL |
Steps
Sampling
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.
Tissue Fixation
Incubate the tissue in 100µL of FAA (see MATERIALS) at Room temperature for 1h 0m 0s.
Wash the tissue successively for 0h 10m 0s at Room temperature with 100µL 70% EtOH, 90% EtOH, and twice with 100% EtOH.
Wash the tissue twice with 100µL 100% MeOH for 0h 10m 0s and leave the tissue in MeOH after the second wash at -20°C 0h 30m 0s.
Tissue Permeabilization
Rehydrate the tissue by 0h 5m 0s successive washes with 100µL 75%, 50%, 25% MeOH in DPBST.
Incubate the tissues in 100µL 1x CWDES (see MATERIALS) On ice for 0h 5m 0s.
| A | B |
|---|---|
| A | B |
| Reagent | Amount |
| 10x CWDES | 10 µL |
| SUPERase IN | 1 µL |
| DPBST | 89 µL |
1x CWDES
Remove the CWDES and add 100µL fresh & cold 1x CWDES. Then, incubate the tissue at Room temperature for 0h 30m 0s.
Wash the tissue twice with 100µL DPBSTR (see MATERIALS).
Fix the tissue by incubating in 100µL 10% (v/v) formaldehyde in DPBST at Room temperature for 0h 30m 0s.
Wash the tissue twice with 100µL DPBSTR at Room temperature (0h 1m 0s each).
Incubate the tissue in 100µL digestion solution at 37°C for 0h 30m 0s.
| A | B |
|---|---|
| Reagent | Amount |
| Proteinase K buffer (see MATERIALS) | 99 µL |
| Proteinase K | 1 µL |
Digestion solution
Wash the tissue twice with 100µL DPBSTR at Room temperature (0h 1m 0s each).
Fix the tissue by incubating in 100µL 10% (v/v) formaldehyde in DPBST at Room temperature for 0h 30m 0s.
Wash the tissue twice with 100µL DPBSTR at Room temperature (0h 5m 0s each).
Gene Specific Probe Hybridization
Mix gene specific probes at the concentration of 5 nM per oligo.
Heat the probe mixture at 90°C for 0h 3m 0s and let it cool down at Room temperature.
Incubate the tissue in the hybridization mixture at 40°C for 3h 0m 0s or 3h 0m 0s
| A | B |
|---|---|
| Reagent | Amount |
| 20xSSC | 10 µL |
| Formamide | 30 µL |
| 10% Triton-X | 10 µL |
| 200 mM RVC | 10 µL |
| SUPERase IN | 1 µL |
| Probe mix (500 nM per oligo) | 2 µL |
| Nuclease Free Water | 37 µL |
Hybridization mixture
Wash the tissue twice with 100µL DPBSTR at 37°C for 0h 30m 0s.
Wash the tissue twice with 100µL 4xSSC in DPBSTR at 37°C for 0h 30m 0s.
| A | B |
|---|---|
| Reagent | Amount |
| 20xSSC | 20 µL |
| DPBST | 79 µL |
| SUPERase IN | 1 µL |
4xSSC in DPBSTR
Rinse the tissue with 100µL DPBSTR at Room temperature.
Ligation
Incubate the tissue in 100µL ligation mixture WITHOUT ligase On ice for 0h 5m 0s.
| A | B |
|---|---|
| Reagent | Amount |
| 10x ligation buffer | 10 µL |
| BSA (2mg/ml) | 0.5 µL |
| SUPERase IN | 1 µL |
| Nuclease Free Water | 88.5 µL |
Ligation mixture without ligase
Incubate the tissue in 100µL ligation mixture WITH ligase at Room temperature .
| A | B |
|---|---|
| Reagent | Amount |
| 10x ligation buffer | 10 µL |
| BSA (20 mg/ml) | 0.5 µL |
| SUPERase IN | 1 µL |
| T4 DNA ligase | 2 µL |
| Nuclease Free Water | 86.5 µL |
Ligation mixture
Rolling Circle Amplification (RCA)
Wash the tissue with 100µL DPBSTR for 0h 5m 0s.
Incubate the tissue in 100µL RCA mixture WITHOUT equiPhi29 DNA polymerase On ice for 0h 5m 0s.
| A | B |
|---|---|
| Reagent | Amount |
| 10x equiPhi29 DNA polymerase buffer | 10 µL |
| 10 mM dNTP | 2.5 µL |
| 4 mM aminoallyl-dUTP | 0.5 µL |
| BSA (20 mg/ml) | 0.5 µL |
| DTT (100 mM) | 1 µL |
| SUPERase IN | 1 µL |
| Nuclease Free Water | 84.5 µL |
RCA mixture without equiPhi29 DNA polymerase
Incubate the tissue in 100µL RCA mixture WITH equiPhi29 DNA polymerase at 37°C 0h 5m 0s.
| A | B |
|---|---|
| Reagent | Amount |
| 10x equiPhi29 DNA polymerase buffer | 10 µL |
| 10 mM dNTP | 2.5 µL |
| 4 mM aminoallyl-dUTP | 0.5 µL |
| BSA (20 mg/ml) | 0.5 µL |
| DTT (100 mM) | 1 µL |
| SUPERase IN | 1 µL |
| equiPhi29 DNA polymerase | 5 µL |
| Nuclease Free Water | 79.5 µL |
RCA mixture with equiPhi29 DNA polymerase
Wash the tissue twice with 100µL DPBSTR for 0h 10m 0s.
Post-Amplification Fixation
Incubate the tissue in 100µL BS(PEG9) solution at Room temperature for 1h 0m 0s.
| A | B |
|---|---|
| Reagent | Amount |
| DPBST | 98 µL |
| BS(PEG9) stock | 2 µL |
BS(PEG9) solution
Aspire BS(PEG9) solution and incubate the tissue in 100µL 1 M Tris-HCl pH 8.0 at Room temperature for 0h 30m 0s.
Rinse the tissue in 100µL DPBST.
Gel Embedding
Incubate the tissue in 100µL monomer solution On ice for 0h 30m 0s.
| A | B |
|---|---|
| Reagent | Amount |
| 20% acrylamide | 20 µL |
| 2% bis-acrylamide | 10 µL |
| 2xSSC | 70 µL |
Monomer solution
Aspire the monomer solution and add 50µL 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 1h 0m 0s––2h 0m 0s until the gel solidifies.
| A | B |
|---|---|
| Reagent | Amount |
| Monomer solution | 48.9 µL |
| 10% APS stock | 1 µL |
| TEMED | 0.1 µL |
Gelling solution
Carefully remove the coverslip with forceps, and wash the tissue with 100µL DPBST for 0h 5m 0s.
Incubate the tissue in ClearSee until imaging.
Target Detection with Sequence-By-Hybridization
Wash the tissue twice with100µL 2xSSC for 0h 1m 0s.
Incubate the tissue in 100µL Bridge probe mixture at Room temperature for 1h 0m 0s.
| A | B |
|---|---|
| Reagent | Amount |
| 2x hybridization buffer | 50 µL |
| Bridge probes (10uM stock) | 1 µL each (typically 4 probes) |
| Nuclease free water | up to 100 µL |
Bridge probe mixture
Wash the tissue twice with 100µL 2xSSC for 0h 1m 0s.
Incubate the tissue in 100µL Detection probe mixture at Room temperature for 1h 0m 0s.
| A | B |
|---|---|
| Reagent | Amount |
| 2x hybridization buffer | 50 µL |
| Detection probes (10uM stock) | 1 µL each (4 probes) |
| Calcofluor White | 1 µL |
| Nuclease free water | up to 100 µL |
Detection probe mixture
Wash the tissue twice with 100µL 2xSSC for 0h 1m 0s.
Wash the tissue with 100µL ClearSee, and keep it in ClearSee for imaging.
Imaging with a confocal microscope.
After the imaging, strip the bridge/detection probes by incubating the tissue in100µL stripping solution (65% formamide in 2xSSC) at 30°C for 0h 30m 0s.
| A | B |
|---|---|
| Reagent | Amount |
| Formamide | 65 µL |
| 20xSSC | 10 µL |
| Nuclease free water | up to 100 µL |
Stripping solution
Go to Step 35 for the next round of imaging.
