PHYTOMap experimental procedure

The following chemicals and enzymes were used: a poly-d-lysine coated dish (MatTek, catalogue no. P35GC-1.5-14-C); T4 DNA ligase (Thermo Fisher Scientific, catalogue no. EL0011); EquiPhi29 DNA polymerase (Thermo Fisher Scientific, catalogue no. A39391); SUPERaseIn RNase inhibitor (Invitrogen, catalogue no. AM2696); aminoallyl dUTP (AnaSpec, catalogue no. AS-83203); Dulbecco’s phosphate-buffered saline (DPBS) (Sigma, catalogue no. D8662); molecular biology grade BSA (New England Biolabs, catalogue no. B9000S); dNTPs (New England Biolabs, catalogue no. N0447S); Fluorescent Brightener 28 disodium salt solution (Sigma, catalogue no. 910090); formaldehyde solution for molecular biology, 36.5%–38% in water (Sigma, catalogue no. F8775); Triton-X (Sigma, catalogue no. 93443); Proteinase K (Invitrogen, catalogue no. 25530049); nuclease-free water (Invitrogen, catalogue no. AM9937); BS(PEG)9 (Thermo Fisher Scientific, catalogue no. 21582); 20× SSC buffer (Sigma-Aldrich, catalogue no. S6639); ribonucleoside vanadyl complex (New England Biolabs, catalogue no. S1402S); formamide (Sigma, catalogue no. F9037); RNase-free Tris buffer pH 8.0 (Invitrogen, catalogue no. AM9855G); RNase-free EDTA pH 8.0 (Invitrogen, catalogue no. AM9260G); cellulase (Yaklut, catalogue no. YAKL0013); macerozyme (Yakult, catalogue no. YAKL0021); and pectinase (Thermo Fisher Scientific, catalogue no. ICN19897901).

Target genes were selected manually based on their cell type-specific expression. Probes were constructed by combining the probe design used in STARmap²⁴ and HYBISS¹³ (Extended Data Fig. ​Fig.1a).1a). A SNAIL probe—a pair comprising a padlock probe (PLP) and a primer—was designed. (1) For each gene, 40–50-nucloetide sequences with a GC content of 40%–60% were selected and it was confirmed that there was no homologous region in the other transcripts by blasting against TAIR10 Arabidopsis genome. (2) Selected sequences were split into halves, each of 20–25 nucleotides (the 5′ halves for PLPs and the 3′ halves for primers), with a two-nucleotide gap between, ensuring that the melting temperature (T_m) of each half is around 60 °C. (3) PLPs have complementary sequences for target specific bridge probes. (4) Four SNAIL probes were designed for each gene. (5) PLPs and primers have complementary sequences to form a circular structure. Bridge probes and detection read-out probes were designed as described previously¹³ and detailed in Supplementary Table 2. All probes were manufactured by Integrated DNA Technologies. SNAIL probes were manufactured in the form of oPools Oligo Pools with desalting purification. Bridge probes were manufactured individually with desalting purification. Detection read-out probes were manufactured individually with HPLC purification.

Five-day-old root tips were cut on the agar plate using a razor blade, mounted on a dry poly-d-lysine coated dish using tweezers, and immediately fixed, dehydrated and rehydrated in a manner similar to that described in previous studies^4,15 with modifications. The following steps were conducted on the dish. Arabidopsis root tips were immersed in FAA (16% v/v formaldehyde, 5% v/v acetic acid and 50% ethanol) for 1 h at room temperature. RNase-free water was used throughout the entire protocol. Samples were then dehydrated in a series of 10-min washes once in 70% (v/v in nuclease-free water) ethanol, once in 90% ethanol and twice in 100% ethanol, followed by two 10-min washes in 100% methanol, and then were stored in 100% methanol at −20 °C overnight. The next day, samples were rehydrated in a series of 5-min washes in 75% (v/v), 50% and 25% methanol in DPBS-T (0.1% Tween 20 in DPBS) at room temperature. The cell wall was partially digested by incubating samples in cell wall digestion solution (0.06% cellulase, 0.06% macerozyme, 0.1% pectinase, and 1% SUPERase in DPBS-T) for 5 min on ice, and then for 30 min at room temperature. After two washes in DPBS-TR (DPBS-T and 1% SUPERase), samples were fixed in 10% (v/v) formaldehyde for 30 min at room temperature and washed with DPBS-TR. Proteins were digested by incubating samples in protein digestion buffer (0.1 M Tris–HCl pH 8, 50 mM EDTA pH 8) with a 1:100 volume of Proteinase K (20 mg ml⁻¹, RNA grade; Invitrogen, catalogue no. 25530049) for 30 min at 37 °C. After two washes in DPBS-TR, samples were fixed in 10% (v/v) formaldehyde for 30 min at room temperature and washed with DPBS-TR.

The following steps are based on STARmap protocols²⁴ with modifications. A pool of SNAIL probes (500 nM each) was heated at 90 °C for 5 min and cooled at room temperature. Samples were incubated in hybridization buffer (2× SSC, 30% formamide, 1% Triton-X, 20 mM ribonucleoside vanadyl complex and pooled SNAIL probes at 10 nM per oligo) in a 40 °C humidified oven overnight. After hybridization, samples were washed twice in DPBS-TR and once in 4× SSC in DPBS-TR for 30 min at 37 °C and rinsed with DPBS-TR at room temperature. Samples were then incubated in a T4 DNA ligation mixture (1:50 dilution of T4 DNA ligase supplemented with 1× BSA and 0.2 U µl⁻¹ of SUPERase-In) at room temperature overnight. After ligation, samples were washed twice with DPBS-TR for 10 min at room temperature and incubated in a rolling circle amplification (RCA) mixture (1:20 dilution of equiPhi29 DNA polymerase, 250 µM dNTP, 0.1 µg µl⁻¹ BSA, 1 mM dithiothreitol, 0.2 U µl⁻¹ of SUPERase-In and 20 µM aminoallyl dUTP) at 37 °C overnight. After RCA, samples were rinsed in DPBS-T and covalently cross-linked with 4.3 µg µl⁻¹ BS(PEG)9 in DPBS-T. BS(PEG)9 was then quenched by incubating samples in 1 M Tris–HCl (pH 8) for 30 min at room temperature.

After the fixation of DNA amplicons, samples were embedded in acrylamide gel by incubating in a polymerization mixture (4% acrylamide, 0.2% bis-acrylamide, 0.1% ammonium persulfate and 0.1% tetramethylethylenediamine in DPBS-T) for 1.5 h at room temperature. Samples were then rinsed in DPBS-T. After gel embedding, samples were cleared by incubating in ClearSee²⁵ at room temperature overnight.

Samples were washed with 2× SSC for 5 min at room temperature and then incubated in a bridge probe hybridization mixture (2× SSC, 20% formamide and four bridge probes at 100 nM per oligo in water) for 1 h at room temperature. After washing twice in 2× SSC for 5 min at room temperature, samples were incubated in a detection probe hybridization mixture (2× SSC, 20% formamide, 1:100 dilution of Calcofluor White (Fluorescent Brightener 28 disodium salt solution) and fluorescent detection oligos at 100 nM per oligos in water) for 1 h at room temperature. Samples were washed in 2× SSC and ClearSee for 5 min at room temperature and stored in ClearSee until imaging. After imaging, the PHYTOMap signal was stripped by incubating in stripping buffer (65% formamide in 2× SSC) at 30 °C for 30 min.

Imaging was performed using a Leica Stellaris 8 confocal microscope equipped with a DMi8 CS Premium, supercontinuum white light laser, laser 405 DMOD, power HyD detectors and an HC PL APO CS2 ×40/1.10 water objective. The image size for a field-of-view was 512 × 512 pixels with a voxel size of 0.57 μm × 0.57 μm × 0.42 μm, and three fields-of-view were acquired for each root sample unless otherwise stated. The 2D images shown in Extended Data Fig. ​Fig.4b4b were taken in a scan format of 2,048 × 2,048 pixels with denoising (averaging two images). The following channel settings were used: 405 nm excitation, 420–510 nm emission; 499 nm excitation, 504–554 nm emission; 554 nm excitation, 559–650 nm emission; 649 nm excitation, 657–735 nm emission; 752 nm excitation, 760–839 nm emission.

Arabidopsis plants were grown in soil for 20 days with a 12 h light period. The fifth leaf (the largest) was used for the experiment. Leaves were processed as described above with slight modifications. Because the whole-mount leaf did not attach to the poly-d-lysine coated dish, the tissue was fixed in a 1.5 ml tube with FAA. A vacuum was applied to facilitate fixation. After the first fixation, the tissue was transferred to a poly-d-lysine coated dish and the downstream steps were carried out on the dish. The tissue was not embedded in the gel, because we did not perform multiple rounds of imaging. Before imaging, the tissue was mounted on a glass slide with a coverslip on top to immobilize the tissue. SNAIL probes for UBQ10 (AT4G05320) were used (Supplementary Table 2).

The cost of PHYTOMap experiments is approximately US$80 for a 28-gene experiment and US$230 for a 96-gene experiment (Supplementary Fig. 3b and detailed in Supplementary Table 3), where each experiment can accommodate five or more root tips, which can be from different treatments and/or genotypes. The initial investment (reagent cost) to set up PHYTOMap experiments is approximately US$2,700 and US$5,500 for a 28-gene and 96-gene experiment, respectively.