May 24, 2023

Public workspacePHYTOMap in Arabidopsis root tips

DOI
dx.doi.org/10.17504/protocols.io.rm7vzbp4xvx1/v1
  • 1The Salk Institute for Biological Studies;
  • 2Howard Hughes Medical Institute
Tatsuya Nobori
Open access
DOI: dx.doi.org/10.17504/protocols.io.rm7vzbp4xvx1/v1
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 28, 2023
Last Modified: May 24, 2023
Protocol Integer ID: 81174
Keywords: In situ hybridization, plant biology, spatial transcriptomics
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
AB
ReagentAmount
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
AB
ReagentAmount
32% formaldehyde450 µL
Acetic acid50 µL
Ethanol500 µL


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.


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
AB
ReagentAmount
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
AB
ReagentAmount
32% formaldehyde450 µL
Acetic acid50 µL
Ethanol500 µL
Safety warnings
Attention
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
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
Tissue Fixation
Incubate the tissue in Amount100 µL of FAA (see MATERIALS) at TemperatureRoom temperature for Duration01:00:00 .

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 Duration00:10:00 at TemperatureRoom temperature with Amount100 µL 70% EtOH, 90% EtOH, and twice with 100% EtOH.

10m
Wash the tissue twice with Amount100 µL 100% MeOH for Duration00:10:00 and leave the tissue in MeOH after the second wash at Temperature-20 °C DurationOvernight .

40m
Tissue Permeabilization
Tissue Permeabilization

Rehydrate the tissue by Duration00:05:00 successive washes with Amount100 µL 75%, 50%, 25% MeOH in DPBST.
5m
Incubate the tissues in Amount100 µL 1x CWDES (see MATERIALS) TemperatureOn ice for Duration00:05:00 .

AB
ReagentAmount
10x CWDES10 µL
SUPERase IN1 µL
DPBST89 µL
1x CWDES
5m
Remove the CWDES and add Amount100 µL fresh & cold 1x CWDES. Then, incubate the tissue at TemperatureRoom temperature for Duration00:30:00 .
30m
Wash the tissue twice with Amount100 µL DPBSTR (see MATERIALS).

Fix the tissue by incubating in Amount100 µL 10% (v/v) formaldehyde in DPBST at TemperatureRoom temperature for Duration00:30:00 .

30m
Wash the tissue twice with Amount100 µL DPBSTR at TemperatureRoom temperature (Duration00:01:00 each).

1m
Incubate the tissue in Amount100 µL digestion solution at Temperature37 °C for Duration00:30:00 .

AB
ReagentAmount
Proteinase K buffer (see MATERIALS)99 µL
Proteinase K1 µL
Digestion solution

30m
Wash the tissue twice with Amount100 µL DPBSTR at TemperatureRoom temperature (Duration00:01:00 each).

1m
Fix the tissue by incubating in Amount100 µL 10% (v/v) formaldehyde in DPBST at TemperatureRoom temperature for Duration00:30:00 .

30m
Wash the tissue twice with Amount100 µL DPBSTR at TemperatureRoom temperature (Duration00:05:00 each).

5m
Gene Specific Probe Hybridization
Gene Specific Probe Hybridization
Mix gene specific probes at the concentration of 5 nM per oligo.
Heat the probe mixture at Temperature90 °C for Duration00:03:00 and let it cool down at TemperatureRoom temperature .

3m
Incubate the tissue in the hybridization mixture at Temperature40 °C for Duration03:00:00 or DurationOvernight

AB
ReagentAmount
20xSSC10 µL
Formamide30 µL
10% Triton-X10 µL
200 mM RVC10 µL
SUPERase IN1 µL
Probe mix (500 nM per oligo)2 µL
Nuclease Free Water 37 µL
Hybridization mixture

Note
The design of probes is described in our paper.

6h
Wash the tissue twice with Amount100 µL DPBSTR at Temperature37 °C for Duration00:30:00 .

30m
Wash the tissue twice with Amount100 µL 4xSSC in DPBSTR at Temperature37 °C for Duration00:30:00 .

AB
ReagentAmount
20xSSC20 µL
DPBST79 µL
SUPERase IN1 µL
4xSSC in DPBSTR

30m
Rinse the tissue with Amount100 µL DPBSTR at TemperatureRoom temperature .

Ligation
Ligation
Incubate the tissue in Amount100 µL ligation mixture WITHOUT ligase TemperatureOn ice for Duration00:05:00 .

AB
ReagentAmount
10x ligation buffer10 µL
BSA (2mg/ml)0.5 µL
SUPERase IN1 µL
Nuclease Free Water 88.5 µL
Ligation mixture without ligase

5m
Incubate the tissue in Amount100 µL ligation mixture WITH ligase at TemperatureRoom temperature DurationOvernight .
AB
ReagentAmount
10x ligation buffer10 µL
BSA (20 mg/ml)0.5 µL
SUPERase IN1 µL
T4 DNA ligase2 µL
Nuclease Free Water 86.5 µL
Ligation mixture
Rolling Circle Amplification (RCA)
Rolling Circle Amplification (RCA)
Wash the tissue with Amount100 µL DPBSTR for Duration00:05:00 .

5m
Incubate the tissue in Amount100 µL RCA mixture WITHOUT equiPhi29 DNA polymerase TemperatureOn ice for Duration00:05:00 .

AB
ReagentAmount
10x equiPhi29 DNA polymerase buffer10 µL
10 mM dNTP2.5 µL
4 mM aminoallyl-dUTP0.5 µL
BSA (20 mg/ml)0.5 µL
DTT (100 mM)1 µL
SUPERase IN1 µL
Nuclease Free Water 84.5 µL
RCA mixture without equiPhi29 DNA polymerase



5m
Incubate the tissue in Amount100 µL RCA mixture WITH equiPhi29 DNA polymerase at Temperature37 °C DurationOvernight .

AB
ReagentAmount
10x equiPhi29 DNA polymerase buffer10 µL
10 mM dNTP2.5 µL
4 mM aminoallyl-dUTP0.5 µL
BSA (20 mg/ml)0.5 µL
DTT (100 mM)1 µL
SUPERase IN1 µL
equiPhi29 DNA polymerase5 µL
Nuclease Free Water 79.5 µL
RCA mixture with equiPhi29 DNA polymerase

5m
Wash the tissue twice with Amount100 µL DPBSTR for Duration00:10:00 .

10m
Post-Amplification Fixation
Post-Amplification Fixation
Incubate the tissue in Amount100 µL BS(PEG9) solution at TemperatureRoom temperature for Duration01:00:00 .
AB
ReagentAmount
DPBST98 µL
BS(PEG9) stock2 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 Amount100 µL 1 M Tris-HCl pH 8.0 at TemperatureRoom temperature for Duration00:30:00 .

30m
Rinse the tissue in Amount100 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
Gel Embedding
Incubate the tissue in Amount100 µL monomer solution TemperatureOn ice for Duration00:30:00 .
AB
ReagentAmount
20% acrylamide20 µL
2% bis-acrylamide10 µL
2xSSC70 µL
Monomer solution
30m
Aspire the monomer solution and add Amount50 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 TemperatureRoom temperature for Duration01:00:00 ––Duration02:00:00 until the gel solidifies.
AB
ReagentAmount
Monomer solution48.9 µL
10% APS stock1 µL
TEMED0.1 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 Amount100 µL DPBST for Duration00:05:00 .

5m
Incubate the tissue in ClearSee until imaging.
Target Detection with Sequence-By-Hybridization
Target Detection with Sequence-By-Hybridization
Wash the tissue twice withAmount100 µL 2xSSC for Duration00:01:00 .

1m
Incubate the tissue in Amount100 µL Bridge probe mixture at TemperatureRoom temperature for Duration01:00:00 .
AB
ReagentAmount
2x hybridization buffer50 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 Amount100 µL 2xSSC for Duration00:01:00 .

1m
Incubate the tissue in Amount100 µL Detection probe mixture at TemperatureRoom temperature for Duration01:00:00 .
AB
ReagentAmount
2x hybridization buffer50 µL
Detection probes (10uM stock) 1 µL each (4 probes)
Calcofluor White1 µL
Nuclease free water up to 100 µL
Detection probe mixture
1h
Wash the tissue twice with Amount100 µL 2xSSC for Duration00:01:00 .

1m
Wash the tissue with Amount100 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 inAmount100 µL stripping solution (65% formamide in 2xSSC) at Temperature30 °C for Duration00:30:00 .
AB
ReagentAmount
Formamide65 µL
20xSSC10 µL
Nuclease free water up to 100 µL
Stripping solution
30m
Go to Step 35 for the next round of imaging.