Discovery of FoTO1 and Taxol genes allows biosynthesis of baccatin III

Chemical and organic supplies

Chemical requirements have been bought from the next distributors (with catalogue quantity listed): taxusin (TargetMol; TN6763), 1-hydroxybaccatin I (LKT Labs; T0092), baccatin VI (Santa Cruz Biotechnology; sc-503244), 10-deacetylbaccatin III (Sigma-Aldrich; D3676), baccatin III (MedChemExpress; HY-N6985) and 9-dihydro-13-acetylbaccatin III (TargetMol; T5132). Taxadien-5α-ol was synthesized as beforehand described¹⁸. Taxus media var. hicksii was obtained from FastGrowingTrees.

Tissue preparation and single-nucleus sequencing

The cells of Taxus species, like these of many crops, are sometimes two to a few instances bigger than the 35-µm diameter restrict for the usual 10x Genomics Chromium single-cell library units. Consequently, single-cell isolation approaches (equivalent to protoplasting) risked introducing a extreme cell-type bias, and we as an alternative tailored beforehand described nuclei isolation strategies⁵² right into a conifer-compatible snRNA-seq protocol. Taxus media var. hicksii aerial tissues (needles, stems and bud scales) have been manually disrupted by razor blade and detergent remedy, adopted by DNA staining, fluorescence-activated cell sorting (FACS) purification and library synthesis within the 10x Chromium platform. Nuclei extraction buffer (NIB) consisted of 5 mM MgCl₂, 10 mM HEPES pH 7.6, 0.8 M sucrose, 0.1% Triton X-100 and (for density matching to stop nuclei settling throughout movement sorting) 1% dextran T40 and a pair of% Ficoll. On the day of use, NIB was supplemented with 1 mM dithiothreitol. All nuclei-extraction steps have been carried out at 4 °C and wide-bore pipette suggestions have been used when dealing with nuclei. Steps between tissue assortment and loading into the Chromium machine have been accomplished inside 90 min to keep away from RNA loss. To isolate nuclei, roughly 1 g of T. media tissue was faraway from the plant and instantly positioned in a Petri dish with 10 ml NIB. Tissue was chopped by hand at round 200 rpm with a contemporary razor blade for five min till many of the massive tissue was damaged down, and was then gently rocked at 4 °C for 15 min. To take away massive particles, disrupted tissue was then handed by way of a pre-wet 100-μm cell strainer stacked on prime of a 40-μm cell strainer. Nuclei have been gently pelleted at 300g at 4 °C for five min and resuspended in 1 ml NIB with 5 ng μl⁻¹ 4,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific) and 5 ng μl⁻¹ propidium iodide. Utilizing a Sony SH800 cell sorter with a 70-μm chip, 140,000–200,000 nuclei have been sorted right into a tube containing 1 ml PBS+ (PBS, 0.1% bovine serum albumin and 20 U ml⁻¹ Invitrogen ribonuclease inhibitor). The gating technique is proven in Supplementary Fig. 22. Nuclei have been centrifuged at 300g at 4 °C for five min, then gently resuspended in 40 μl PBS+. Nuclei have been instantly loaded onto a 10x Genomics Chromium controller and libraries have been generated utilizing v3 chemistry. Libraries have been sequenced on an Illumina NextSeq 3000.

Multiplexed tissue elicitation

For the multiplexed elicitation experiment, Taxus needles have been subjected to perturbation in deep-well 96-well plates with 200 μl MS medium (7.5 g l⁻¹ Murashige and Skoog macronutrients (Fisher), 3 g l⁻¹ sucrose, pH 5.7) supplemented with elicitor. Two needles (organic replicates), every from two developmental phases (younger and mature), have been handled with every elicitation situation (17 circumstances listed in Supplementary Desk 2) for every time level (1, 2, 3 and 4 days), leading to 272 tissue samples (2 replicates of 136 perturbations). To attenuate contamination, needles have been washed completely in sterile water earlier than shifting to MS plates, which have been sealed with breathable rayon movie (VWR) and positioned below 18-h gentle cycles. Tissue elicitation was began at staggered instances so that every one tissues may very well be collected concurrently. To extract nuclei from elicited tissues, all tissues have been mixed in a wire mesh, washed with water and subjected to the above nuclei-extraction protocol.

Evaluation of single-cell knowledge

Reads have been cleaned with Trimmomatic⁵³ and mapped to the genomes of T. chinensis⁵ with STARsolo (v.2.7.10b)⁵⁴ (STAR…–runThreadN 32–alignIntronMax 10000–soloUMIlen 12–soloCellFilter EmptyDrops_CR–soloFeatures GeneFull–soloMultiMappers EM–soloType CB_UMI_Simple). Ambient RNA was eliminated with CellBender (v.0.3.0)⁵⁵. Utilizing the doubletdetection (v.4.2) library⁵⁶, doublets have been eliminated, in addition to cells with outlier numbers of reads or through which most reads have been probably the most expressed genes (pct_counts_in_top_20_genes < 25). Genes have been faraway from evaluation if expressed in fewer than 50 cells. For built-in UMAP plots, scVI was used to combine cells from a number of single-cell experiments⁵⁷. Scanpy (v.1.10.1)⁵⁸ was used for processing and plotting post-filtered nuclear transcriptomes. For co-expression evaluation and gene–gene correlation calculations, scVI-normalized⁵⁷ transcriptomes (8,039 elicited transcriptomes, 3,027 naive transcriptomes from younger tissues and 6,077 naive transcriptomes from mature tissues) have been clustered into 2,901 cell states (round 10 cells per state) by Leiden clustering⁵⁸, after which uncooked reads from every cluster have been pooled to yield pseudobulk transcriptomes. These pseudobulk transcriptomes have been used to calculate gene–gene correlations. For module evaluation, uncooked reads have been analysed by a cNMF bundle²⁸ run with default parameters, besides ‘complete modules’) to yield gene modules and their utilization throughout cells. Factorization approximates the noticed dataset because the product of two smaller, significant matrices: (i) a gene–module matrix (a weight worth for every gene in every module); and (ii) a cell–module matrix (expression values of every module in every cell) (Fig. 2c). The load values of the gene–module matrix can be utilized as scores that determine the genes that dominate every module; top-scoring genes from the identical module have coordinated expression patterns and are more likely to be a part of the identical molecular processes. This method adapts to the wealthy however noisy knowledge inherent in single-cell evaluation, and divulges patterns of coordinated gene expression which may not be obvious from linear correlation evaluation. For instance, it permits for genes to be in a number of, overlapping modules, which is more likely to higher symbolize how genes in a extremely branched metabolism could also be expressed. The ‘complete modules’ parameter was scanned from ok = 50 to ok = 400 to find out the sensitivity of the outcomes on this parameter (Supplementary Fig. 1).

Bulk RNA-seq evaluation

Uncooked fastq information from six earlier research^{5,59,60,61,62,63} have been downloaded from NCBI (PRJNA493167, PRJNA251671, PRJNA733140, PRJNA427840, PRJNA497542, PRJNA499080 and PRJNA864083), cleaned with Trimmomatic⁵³ and aligned to the T. chinensis genome⁵ (STAR map⁶⁴). Gene–gene correlation was calculated with numpy. Mutual rank (mr), used to calculate the gene linkage maps (Fig. 1d), is outlined as:

the place rank_ij signifies the Pearson correlation rank of gene i to gene j.

Cloning of Taxus genes

The cloning of cytosolic diterpenoid enhance genes (tHMGR and GGPPS), cytosolic TDS1 and TDS2, T5αH, TAT, T10βΗ, DBAT, T13αΗ and TAX19 genes has been described beforehand^18,65. Candidate genes have been amplified from T. media gDNA or cDNA (generated with SuperScript IV, Thermo Fisher Scientific) by PCR (PrimeStar, Takara Bio R045B, primers in Supplementary Desk 13), and the PCR merchandise have been ligated with AgeI- and XhoI- (New England Biolabs) linearized pEAQ-HT vector⁶⁶ utilizing HiFi DNA meeting combine (New England Biolabs). Gene annotations used for cloning have been taken from the T. chinensis genome⁵ by default, however have been BLAST-searched towards the T. media genome (NCBI PRJNA1136025) to find out whether or not different gene fashions have been accessible. Constructs have been reworked into 10-beta competent E. coli cells (New England Biolabs). Plasmid DNA was remoted utilizing the QIAprep Spin Miniprep equipment (QIAGEN) and the sequence was verified by whole-plasmid sequencing (Plasmidsaurus).

Transient expression of Taxus genes in N. benthamiana by Agrobacterium-mediated infiltration

pEAQ-HT plasmids containing the Taxus gene have been reworked into Agrobacterium tumefaciens (pressure GV3101) cells utilizing the freeze–thaw technique. Remodeled cells have been grown on micro organism screening medium 523-agar (Phytotech Labs) plates containing kanamycin and gentamicin (50 μg ml⁻¹ and 30 μg ml⁻¹, respectively; similar for the 523 medium beneath), at 30 °C for 2 days. Single colonies have been then picked and grown in a single day at 30 °C in 523-kanamycin–gentamicin liquid medium. The in a single day cultures have been used to make dimethyl sulfoxide (DMSO) shares (7% DMSO) for long-term storage within the −80 °C fridge. For routine N. benthamiana infiltration experiments, particular person Agrobacterium DMSO shares have been streaked out on 523-agar containing kanamycin and gentamicin and grown for round one to 2 days at 30 °C. Patches of cells have been scraped off from particular person plates utilizing 10-μl inoculation loops and resuspended in round 1–2 ml of Agrobacterium induction buffer (10 mM MES pH 5.6, 10 mM MgCl₂ and 150 μM acetosyringone; Acros Organics) in particular person 2-ml safe-lock tubes (Eppendorf). The suspensions have been briefly vortexed to homogeneity and incubated at room temperature for two h. The optical density at 600 nm (OD_600 nm) of the person Agrobacterium suspensions was measured, and the ultimate infiltration resolution, through which the OD_600 nm was 0.2 for every pressure (aside from TDS, T7AT and T7dA; OD_600 nm of 0.6, 0.4 and 0.1, respectively), was ready by mixing particular person strains and diluting with the induction buffer. Leaves of four-week-old N. benthamiana have been infiltrated utilizing needleless 1-ml syringes from the abaxial aspect. Every experiment was examined on leaf 6, 7 and eight (numbered by counting from the underside) of the identical N. benthamiana plant, as three organic replicates.

For the reconstitution of pathways that contain TBT, the next modifications have been made to the process above to extend the manufacturing of the specified benzoylated merchandise: N. benthamiana crops have been watered with 2 mM benzoic acid in water (buffered to pH 5.6) a day earlier than Agrobacterium infiltration, 1 mM benzoic acid was added to the induction buffer and the pH was adjusted to five.6 earlier than getting used for the resuspension of Agrobacterium and preparation of the ultimate infiltration resolution.

Phylogenomic evaluation

FoTO1 homologues have been recognized by scanning the Thousand Plant Transcriptome (1KP)⁶⁷, RefSeq crops and Uniprot Viridiplantae databases with jackhmmer³⁶ (command: jackhmmer -o tempout.txt -E 1e-5 -N 4). Hits with better than 40% sequence gaps to the unique question have been discarded. A phylogenetic tree was generated with the remaining protein sequences with FastTree⁶⁸.

Metabolite extraction of N. benthamiana leaves

5 days after Agrobacterium infiltration, N. benthamiana leaf tissue was collected utilizing a leaf disc cutter 1 cm in diameter and positioned inside a 2-ml safe-lock tube (Eppendorf). Every organic replicate consisted of 4 leaf discs from the identical leaf (roughly 40 mg contemporary weight). The leaf discs have been flash-frozen and lyophilized in a single day. Analyses of the extra hydrophobic metabolites (for instance, compounds 1–6) have been completed by GC–MS, and analyses of the extra hydrophilic metabolites (for instance, compounds 4–18) have been completed by liquid chromatography–mass spectrometry (LC–MS). To extract metabolites, ethyl acetate (ACS reagent grade; J.T. Baker) or 75% acetonitrile (high-performance liquid chromatography (HPLC) grade; Fisher Chemical) in 500 μl water was added to every pattern together with one 5-mm chrome steel bead for GC–MS or LC–MS evaluation, respectively. The samples have been homogenized in a ball mill (Retsch MM 400) at 25 Hz for two min. After homogenization, the samples have been centrifuged at 18,200g for 10 min. For GC–MS samples, the supernatants have been transferred to 50-μl glass inserts, positioned in 2 ml vials and subjected to analysed by the GC–MS instrument. For LC–MS samples, the supernatants have been filtered utilizing 96-well hydrophilic PTFE filters with a pore measurement of 0.45 μm (Millipore) and analysed by the LC–MS instrument.

GC–MS evaluation

GC–MS samples have been analysed utilizing an Agilent 7820A fuel chromatography system coupled to an Agilent 5977B single quadrupole mass spectrometer. Information have been collected with Agilent Enhanced MassHunter and analysed by MassHunter Qualitative Evaluation B.07.00. Separation was completed utilizing an Agilent VF-5HT column (30 m × 0.25 mm × 0.1 μm) with a relentless movement price of helium of 1 ml per min. The inlet was set at 280 °C in break up mode with a ten:1 break up ratio. The injection quantity was 1 μl. Oven circumstances have been as follows: begin and maintain at 130 °C for two min, ramp to 250 °C at 8 °C per min, ramp to 310 °C at 10 °C per min and maintain at 310 °C for five min. The post-run situation was set to 320 °C for 3 min. MS knowledge have been collected with a mass vary 50–550 m/z and a scan pace of 1,562 u s⁻¹ after a 4-min solvent delay. The MSD switch line was set to 250 °C, the MS supply was set to 230 °C and the MS Quad was set to 150 °C.

LC–MS evaluation

LC–MS samples have been analysed on both or each of our two devices: (1) an Agilent 1260 HPLC system coupled to an Agilent 6520 Q-TOF mass spectrometer or (2) an Agilent 1290 HPLC system coupled to an Agilent 6546 Q-TOF mass spectrometer. Sometimes, the 6520 system exhibits higher sensitivity for the extra hydrophobic metabolites, equivalent to 4–6, whereas the 6546 system works higher for the extra hydrophilic, extremely modified taxanes. Information have been collected with Agilent MassHunter Workstation Information Acquisition and analysed by MassHunter Qualitative Evaluation 10.0. Separation was completed utilizing a Gemini 5-μm NX-C18 110-Å column (2 × 100 mm; Phenomenex) with a combination of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a relentless movement price of 400 μl per min at room temperature. The injection quantity was 2 μl or 1 μl for the 6520 or the 6546 system, respectively. The next gradient of solvent B was used: 3% 0–1 min, 3%–50% 1–2 min, 50%–97% 2–12 min, 97% 12–14 min, 97%–3% 14–14.5 min and three% 14.5–21 min (6520 system) and three% 0–1 min, 3%–50% 1–5 min, 50%–97% 5–10 min, 97% 10–12 min, 97%–3% 12–12.5 min and three% 12.5–15 min (6546 system). MS knowledge have been collected utilizing electrospray ionization (ESI) in optimistic mode with a mass vary of fifty–1,200 m/z and a price of 1 spectrum per second (6520 system), or Twin AJS ESI in optimistic mode with a mass vary of 100–1,700 m/z and a price of 1 spectrum per second (6546 system). The ionization supply was set as follows: 325 °C fuel temperature, 10 l min⁻¹ drying fuel, 35 psi nebulizer, 3,500 V VCap, 150 V fragmentor, 65 V skimmer and 750 V octupole 1 RF Vpp (6520 system), or 325 °C fuel temperature, 10 l min⁻¹ drying fuel, 20 psi nebulizer, 3,500 V VCap, 150 V fragmentor, 65 V skimmer and 750 V octupole 1 RF Vpp (6546 system). MS/MS fragmentations have been generated utilizing [M+Na]⁺ because the precursor ion and fragmented with a collision vitality of 30 eV until in any other case said.

Quantification of baccatin III (16)

The samples in Fig. 5i have been analysed by an Agilent 1290 HPLC system coupled to an Agilent 6470 triple quadrupole (QQQ) mass spectrometer to precisely quantify the focus of baccatin III. Information have been collected with Agilent MassHunter Workstation Information Acquisition and analysed by MassHunter Quantitative Evaluation 10.1 and Microsoft Excel. Separation was completed utilizing a ZORBAX RRHD Eclipse Plus C18 Column (2.1 × 50 mm, 1.8 µm; Agilent) with a combination of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a relentless movement price of 600 μl per min at 30 °C. The injection quantity was 0.5 μl. The next gradient of solvent B was used: 30% 0–1 min, 30%–100% 1–5 min, 100% 5–6.5 min, 100%–30% 6.5–7 min and 30% 7–8 min. MS knowledge have been collected utilizing AJS ESI in optimistic mode. A number of response monitoring was used to observe the 609.2 to 549.2 ion transition at a collision vitality of 24 eV because the quantifier, and the 609.2 to 427.1 ion transition at a collision vitality of 32 eV because the qualifier. The ionization supply was set as follows: 250 °C fuel temperature, 12 l min⁻¹ drying fuel, 25 psi nebulizer, 300 °C sheath fuel temperature, 12 l min⁻¹ sheath fuel movement, 3,500 V VCap, 0 V nozzle voltage.

Extraction and purification of taxanes from N. benthamiana

Nicotiana benthamiana crops have been infiltrated with the mixtures of biosynthetic genes proven in Supplementary Desk 12 for the purification of taxusin (6), taxusin (6’), 1β-hydroxytaxusin (6-O1) and 15-hydroxy-11(15→1)abeo-taxusin (6-O2). Lyophilized N. benthamiana supplies have been reduce into small items and extracted with 1 l ethyl acetate (ACS reagent grade; J.T. Baker) in a 2-l flask for 48 h at room temperature with fixed stirring. Extracts have been filtered utilizing vacuum filtration and dried utilizing rotary evaporation. Two rounds of chromatography have been used to isolate compounds of curiosity. The chromatography circumstances for every compound are summarized in Supplementary Desk 12. In short, the primary chromatography was carried out utilizing a 7-cm-diameter column loaded with P60 silica gel (SiliCycle) and utilizing hexane (HPLC grade; VWR) and ethyl acetate because the cell phases. The second chromatography was carried out on an automatic Biotage Selekt system with a Biotage Sfär C18 Duo 6-g column utilizing Milli-Q water and acetonitrile because the cell phases. Fractions have been analysed by LC–MS to determine these containing the compound of curiosity. Desired fractions have been pooled and dried utilizing rotary evaporation (first spherical) or lyophilization (second spherical). Purified merchandise have been analysed by NMR.

NMR evaluation of purified compound

CDCl₃ (Acros Organics) was used because the solvent for all NMR samples. ¹H, ¹³C and 2D-NMR spectra have been acquired on a Varian Inova 600-MHz or a Bruker NEO 500-MHz spectrometer at room temperature utilizing VNMRJ 4.2, and the information have been processed and visualized on MestReNova v.14.3.1-31739. Chemical shifts have been reported in ppm downfield from Me₄Si by utilizing the residual solvent (CDCl₃) peak as an inner commonplace (7.26 ppm for the ¹H and 77.16 ppm for the ¹³C chemical shift). Spectra have been analysed and processed utilizing MestReNova v.14.3.1-31739.

Taxane feeding experiments

Taxus genes have been expressed in N. benthamiana leaves utilizing the Agrobacterium-mediated infiltration technique described above. Three days after Agrobacterium infiltration, taxanes (purified 3O2A (4), taxusin (6), 10-deacetylbaccatin III or 9-dihydro-13-acetylbaccatin III (13); until in any other case specified, a 100-μM resolution after diluting with 10 mM DMSO inventory was used) have been fed into the leaves. Roughly 150 μl of resolution was used per leaf to yield a circle with a diameter round 3 cm, which was marked for reference. After 18–24 h, 4 leaf discs have been collected throughout the marked space with a 1-cm diameter cutter, and LC–MS samples have been ready following the strategies described above.

Building of phylogenetic timber

Sequences from the T. chinensis genome have been chosen utilizing Pfam to determine 672 P450s (PF00067), 218 2-ODDs (PF03171) and 195 acyltransferases (PF02458). P450s have been additional filtered to these longer than 300 amino acids (467 P450s). A number of sequence alignment for every household was carried out utilizing Clustal Omega, and the phylogenetic timber have been constructed utilizing the neighbour-joining technique in Geneious Prime (v.2024.0.4) with 100 bootstrap replicates for preliminary evaluation. Arabidopsis thaliana cinnamate 4-hydroxylase (AtC4H, accession NP_180607.1), A. thaliana gibberellin 20-oxidase1 (AtGA20ox1, accession NP_194272.1), and Hordeum vulgare agmatine coumaroyltransferase (HvACT, accession AAO73071.1) have been used as outgroups for the P450, 2-ODD and acyltransferase households, respectively. All analyses have been carried out with default settings until in any other case specified. Consultant genes from main clades of the preliminary analyses and the Taxol biosynthetic genes have been then chosen to assemble the ultimate phylogenetic timber (Prolonged Information Fig. 9) utilizing the neighbour-joining technique with 1,000 bootstrap replicates.

Purification of proteins and binding assays

All proteins have been purified from commonplace pET28a vectors expressed in BL21DE3 cells (New England Biolabs, C2527H). FoTO1 and FoTO1(ΔCterm) have been purified as C-terminal fusions: His6-3×Flag-TEV-mTurq2-GSG-FoTO1. T5αH and TDS have been purified with N-terminal purification tags (His6-3×Flag-TEV-enzyme) with N-terminal sign peptides eliminated (T5αH, 47 amino acids eliminated; TDS2, 60 amino acids eliminated). Proteins have been purified as beforehand described⁶⁹, with post-lysis steps completed at 4 °C. In short, 1 l of cells have been grown to an OD_600 nm of 0.4–0.5, induced with 0.3 mM IPTG and expressed for 16 h in a single day at 18 °C. Cell pellets have been lysed in lysis buffer (0.5 M NaCl, 20 mM HEPES pH 8.0, 0.1% Triton X-100, 1 mg ml⁻¹ lysozyme, HALT protease cocktail (Thermo Fisher Scientific) and 1 μl ml⁻¹ DNAse I (New England Biolabs)) by sonication, clarified by centrifugation for 1 h at 8,000g. Proteins have been purified on pre-equilibrated Ni-NTA beads (New England Biolabs) and exchanged right into a protein storage buffer (10 mM HEPES-KOH pH 8.0, 50 mM KCL, 10% glycerol, 1 mM DTT and 1 mM EDTA). Purified proteins have been quantified by Bradford assay, and SDS–PAGE gels have been used to confirm protein measurement and proper protein focus.

For every multiscale thermophoresis experiment, one protein was first labelled with the NanoTemper His-Tag labelling equipment (RED-tris-NTA v2, MO-L018) for 30 min at room temperature in accordance with reagent protocols. MST experiments have been carried out in PBS with 0.05% Tween-20 with labelled question protein (T5αH- or TDS-labelled) at 100 nM and a titration sequence of goal protein.

Co-IP

Nicotiana benthamiana leaves have been harvested 4 days after infiltration. Leaf tissue was homogenized in liquid nitrogen and resuspended in extraction buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.6% NP-40, 0.6% CHAPS and 1 mM β-mercaptoethanol)⁷⁰. Lysates have been saved on ice and centrifuged at 20,000g for 10 min at 4 °C. The protein content material of the clarified extract was decided by Bradford assay (Abcam, 119216). Ten microlitres of protein-G-coated magnetic beads (Invitrogen, 10003d) have been washed twice in binding buffer (50 mM Na₂HPO₄, 25 mM citric acid, pH 5.0) earlier than a 1-h incubation at room temperature below agitation with 1 μl anti-V5 antibody. Lysates have been incubated with the indicated compounds for 15 min below agitation. Antibody-bound beads have been then washed twice in extraction buffer and incubated for 15 min below agitation at room temperature with lysate comparable to 100 μg of complete protein content material (roughly 40 μl). After incubation, bead complexes have been washed thrice in extraction buffer and combined with LDS pattern buffer (Invitrogen, NP0007) for subsequent evaluation by immunoblotting.

Immunoblotting

Lysates have been separated for 1.5 h at 80 V on a NuPAGE gel (Invitrogen, NP0321) earlier than switch onto a PVDF membrane utilizing a Bio-Rad Trans-Blot Turbo Switch System (Bio-Rad, 1704150). Immunoblots have been incubated with the indicated antibodies (anti-V5 at 1:1,000 and anti-HA-HRP at 1:2,500) for 3 h at room temperature below agitation. Blots have been subsequently washed and incubated with HRP–protein G (Genscript, M00090) for 1 h, then imaged on the iBright FL1500 Imaging System (Invitrogen, a44241). The extraction buffer was tailored from a beforehand printed process⁷⁰.

Reporting abstract

Additional data on analysis design is offered within the Nature Portfolio Reporting Abstract linked to this text.