May 24, 2023
Version 1
PHYTOMap in Arabidopsis root tips V.1
- Tatsuya Nobori1,2,
- Joseph Ecker1,2
- 1The Salk Institute for Biological Studies;
- 2Howard Hughes Medical Institute
- Nobori Lab @TSL
Protocol Citation: Tatsuya Nobori, Joseph Ecker 2023. PHYTOMap in Arabidopsis root tips. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzbp4xvx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 29, 2023
Last Modified: May 24, 2023
Protocol Integer ID: 81174
Keywords: In situ hybridization, plant biology, spatial transcriptomics, phytomap in arabidopsis root tip, cell type marker genes inarabidopsisroot, arabidopsis root tip, seq datasets in complex plant tissue, targeted observation of gene expression map, spatial analysis of gene expression, spatial mapping of marker gene, gene expression map, complex plant tissue, gene expression, cell rna, marker gene, mount plant tissue, multiplexed fluorescencein situhybridization method, plant hybridization, phytomap, cell wall digestion enzyme solution, major cell type, rna, single cell, individual cell, gene, cell, complex responses of individual cell
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 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.
PHYTOMap principles. a, Target mRNA molecules are hybridized by DNA probes (SNAIL probes) that harbor mRNA species-specific barcode sequences (pink bars). The barcode-containing DNA probes are circularized by ligation and amplified in situ by rolling circle amplification (RCA). b, Amplified DNA barcodes are detected by sequence-by-hybridization. Different fluorescent probes target four DNA barcodes for each imaging round. After the imaging, fluorescent probes are stripped away, and another set of four genes is targeted.
[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 |
Guidelines
See the following paper for more information on PHYTOMap.
Tatsuya Nobori, Marina Oliva, Ryan Lister, and Joseph R. Ecker, bioRxiv, 2022
- 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.
Materials
[Materials]
- Poly-D-Lysine coated dish (MatTek, P35GC-1.5-14-C)
- T4 DNA Ligase (Thermo Scientific, EL0011)
- EquiPhi29 DNA Polymerase (Thermo Scientific, A39391)
- SUPERaseIn RNase Inhibitor (Invitrogen, AM2696)
- Aminoallyl dUTP (AnaSpec, AS-83203)
- Dulbecco’s Phosphate Buffered Saline (DPBS) (Sigma, D8662)
- BSA, molecular biology grade (New England Biolabs, B9000S)
- dNTPs (New England Biolabs, N0447S)
- Fluorescent Brightener 28 disodium salt solution (Sigma, 910090)
- Formaldehyde Solution for Molecular Biology, 36.5-38% in Water (Sigma, F8775)
- Triton-X (Sigma, 93443)
- Proteinase K (Invitrogen, 25530049)
- Nuclease-Free Water (Invitrogen, AM9937)
- BS(PEG)9 (Thermo Scientific, 21582)
- 20 ×SSC buffer (Sigma-Aldrich, S6639)
- Ribonucleoside vanadyl complex (RVC) (New England Biolabs, S1402S)
- Formamide (Sigma, F9037)
- Tris, pH 8.0, RNase-free (Invitrogen, AM9855G)
- EDTA, pH 8.0, RNase-free (Invitrogen, AM9260G)
- Gel Slick™ Solution (Lonza, 50640)
- Cellulase (Yaklut, YAKL0013)
- Macerozyme (Yakult, YAKL0021)
- Pectinase (Fisher Scientific, ICN19897901)
[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 |
Troubleshooting
Safety warnings
See safety data sheets for proper chemical handling, precautionary measures and waste disposal.
Obey all local regulations/guidelines for handling and disposal of used reagents and and solutions containing reagents mixed in.
Formamide:
Handle with proper attire including gloves and eye protection. Work under fume hood when handling solution and dispose of waste appropriately.
Suspected of causing cancer.
May damage fertility or the unborn child.
May cause damage to organs (Blood) through prolonged or repeatedexposure if swallowed.
Formaldehyde:
Handle with proper attire including gloves and eye protection. Work under fume hood when handling solution and dispose of waste appropriately.
May cause cancer.
Toxic if swallowed, in contact with skin or if inhaled.
Causes severe skin burns and eye damage.
May cause an allergic skin reaction.
May cause respiratory irritation.
Suspected of causing genetic defects.
Causes damage to organs (Eyes).
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.
2m
Tissue Fixation
Incubate the tissue in 100 µL of FAA (see MATERIALS) at Room temperature for 01:00:00 .
Note
If most of the root tips detach from the dish, it is necessary to optimize the mounting process for the samples.
1h
Wash the tissue successively for 00:10:00 at Room temperature with 100 µL 70% EtOH, 90% EtOH, and twice with 100% EtOH.
10m
Wash the tissue twice with 100 µL 100% MeOH for 00:10:00 and leave the tissue in MeOH after the second wash at -20 °C Overnight .
40m
Tissue Permeabilization
Rehydrate the tissue by 00:05:00 successive washes with 100 µL 75%, 50%, 25% MeOH in DPBST.
5m
Incubate the tissues in 100 µL 1x CWDES (see MATERIALS) On ice for 00:05:00 .
| A | B | |
| Reagent | Amount | |
| 10x CWDES | 10 µL | |
| SUPERase IN | 1 µL | |
| DPBST | 89 µL |
1x CWDES
5m
Remove the CWDES and add 100 µL fresh & cold 1x CWDES. Then, incubate the tissue at Room temperature for 00:30:00 .
30m
Wash the tissue twice with 100 µL DPBSTR (see MATERIALS).
Fix the tissue by incubating in 100 µL 10% (v/v) formaldehyde in DPBST at Room temperature for 00:30:00 .
30m
Wash the tissue twice with 100 µL DPBSTR at Room temperature (00:01:00 each).
1m
Incubate the tissue in 100 µL digestion solution at 37 °C for 00:30:00 .
| A | B | |
| Reagent | Amount | |
| Proteinase K buffer (see MATERIALS) | 99 µL | |
| Proteinase K | 1 µL |
Digestion solution
30m
Wash the tissue twice with 100 µL DPBSTR at Room temperature (00:01:00 each).
1m
Fix the tissue by incubating in 100 µL 10% (v/v) formaldehyde in DPBST at Room temperature for 00:30:00 .
30m
Wash the tissue twice with 100 µL DPBSTR at Room temperature (00:05:00 each).
5m
Gene Specific Probe Hybridization
Mix gene specific probes at the concentration of 5 nM per oligo.
Heat the probe mixture at 90 °C for 00:03:00 and let it cool down at Room temperature .
3m
Incubate the tissue in the hybridization mixture at 40 °C for 03:00:00 or Overnight
| 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
Note
6h
Wash the tissue twice with 100 µL DPBSTR at 37 °C for 00:30:00 .
30m
Wash the tissue twice with 100 µL 4xSSC in DPBSTR at 37 °C for 00:30:00 .
| A | B | |
| Reagent | Amount | |
| 20xSSC | 20 µL | |
| DPBST | 79 µL | |
| SUPERase IN | 1 µL |
4xSSC in DPBSTR
30m
Rinse the tissue with 100 µL DPBSTR at Room temperature .
Ligation
Incubate the tissue in 100 µL ligation mixture WITHOUT ligase On ice for 00:05:00 .
| 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
5m
Incubate the tissue in 100 µL ligation mixture WITH ligase at Room temperature Overnight .
| 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 100 µL DPBSTR for 00:05:00 .
5m
Incubate the tissue in 100 µL RCA mixture WITHOUT equiPhi29 DNA polymerase On ice for 00:05:00 .
| 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
5m
Incubate the tissue in 100 µL RCA mixture WITH equiPhi29 DNA polymerase at 37 °C Overnight .
| 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
5m
Wash the tissue twice with 100 µL DPBSTR for 00:10:00 .
10m
Post-Amplification Fixation
Incubate the tissue in 100 µL BS(PEG9) solution at Room temperature for 01:00:00 .
| A | B | |
| Reagent | Amount | |
| DPBST | 98 µL | |
| BS(PEG9) stock | 2 µL |
BS(PEG9) solution
Note
BS(PEG9) stock is made by adding 465 µL DMSO into a vial of 100 mg BS(PEG9) and is stored desiccated at -20ºC.
1h
Aspire BS(PEG9) solution and incubate the tissue in 100 µL 1 M Tris-HCl pH 8.0 at Room temperature for 00:30:00 .
30m
Rinse the tissue in 100 µL DPBST.
Note
If multiple rounds of imaging are not necessary, gel embedding may be skipped. However, it is important to note that without gel embedding, there is a higher risk of tissue detachment or movement between imaging rounds. Therefore, it is recommended to carefully handle the tissue.
Gel Embedding
Incubate the tissue in 100 µL monomer solution On ice for 00:30:00 .
| A | B | |
| Reagent | Amount | |
| 20% acrylamide | 20 µL | |
| 2% bis-acrylamide | 10 µL | |
| 2xSSC | 70 µL |
Monomer solution
30m
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 01:00:00 ––02:00:00 until the gel solidifies.
| A | B | |
| Reagent | Amount | |
| Monomer solution | 48.9 µL | |
| 10% APS stock | 1 µL | |
| TEMED | 0.1 µL |
Gelling solution
Note
Coating a coverslip with Gel Slick:
Add Gel Slick Solution onto the coverslip, then wipe gently with a Kimwipe to spread the Gel Slick Solution.
3h
Carefully remove the coverslip with forceps, and wash the tissue with 100 µL DPBST for 00:05:00 .
5m
Incubate the tissue in ClearSee until imaging.
Target Detection with Sequence-By-Hybridization
Wash the tissue twice with100 µL 2xSSC for 00:01:00 .
1m
Incubate the tissue in 100 µL Bridge probe mixture at Room temperature for 01:00:00 .
| 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
1h
Wash the tissue twice with 100 µL 2xSSC for 00:01:00 .
1m
Incubate the tissue in 100 µL Detection probe mixture at Room temperature for 01:00:00 .
| 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
1h
Wash the tissue twice with 100 µL 2xSSC for 00:01:00 .
1m
Wash the tissue with 100 µL 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 in100 µL stripping solution (65% formamide in 2xSSC) at 30 °C for 00:30:00 .
| A | B | |
| Reagent | Amount | |
| Formamide | 65 µL | |
| 20xSSC | 10 µL | |
| Nuclease free water | up to 100 µL |
Stripping solution
30m
Go to Step 35 for the next round of imaging.