Characterization of GO
GO ready by Hummers’ technique was obtained from Graphenea (Spain). The total physicochemical characterization of the GO suspensions in H2O and E3 medium has been beforehand reported11. Right here we offer info on GO suspensions in H2O and completely different cell tradition media, that’s, Dulbecco’s modified Eagle medium (DMEM) (used for the HT-29 cell line) and Roswell Park Memorial Institute (RPMI) (used for main macrophages). TEM and atomic drive microscopy was carried out as beforehand described11. Briefly, carbon-film-coated grids had been pretreated with a glow discharge utilizing a present of −25 mA and for 30 min length. Ten microlitres of the options at 50 µg ml–1 had been deposited on the grid, eradicating extra pattern after 2 min of deposition time. TEM photos had been acquired at 80 kV, and the dimensions distribution evaluation was decided utilizing ImageJ software program model 1.5. Atomic drive microscopy photos had been acquired utilizing a Bruker Multimode 8 atomic drive microscope within the tapping mode with an OTESPA probe.
GO suspensions had been evaluated for endotoxin content material through the use of the TNF-α expression take a look at based mostly on main human monocyte-derived macrophages, as beforehand described31. The cells had been remoted from buffy coats obtained from the Karolinska College Hospital. The samples are utterly anonymized, and the info can’t be traced again to the person donors. Human monocyte-derived macrophages had been uncovered to GO (5–75 μg ml–1) for twenty-four h and cell viability was evaluated utilizing the alamarBlue assay (ThermoFisher Scientific)32. Human monocyte-derived macrophages had been then incubated for twenty-four h with GO at 25 μg ml–1 within the presence or absence of the lipopolysaccharide inhibitor polymyxin B (10 μM). Lipopolysaccharide (0.01 µg ml–1) was used as a optimistic management. Cell tradition supernatants had been collected and TNF-α ranges had been decided by enzyme-linked immunosorbent assay (MabTech). A typical curve was established based mostly on lipopolysaccharide-induced TNF-α.
AhR reporter cells
HT-29-Lucia AhR reporter cells established from the human HT-29 colon adenocarcinoma cell line had been obtained from InVivoGen. The cells had been initially cultured in DMEM supplemented with 4.5 g l–1 glucose, 2 mM l-glutamine, 10% foetal bovine serum (FBS), 100 U ml–1 penicillin, 100 μg ml–1 streptomycin and 100 μg ml–1 Normocin. Following at the least two passages, the expansion medium was supplemented with Zeocin (100 µg ml–1). For the AhR exercise assay, cells had been indifferent with trypsin, centrifuged and resuspended within the take a look at medium with out Normocin and Zeocin at a density of two.8 × 105 cells ml–1. The cells had been uncovered to AA, BA or PA (Sigma-Aldrich); FICZ (Sigma-Aldrich) (200 nM) was used as a optimistic management. Then, cell supernatants had been transferred right into a 96-well plate, and QUANTI-Luc assay answer was added. The measurements had been recorded utilizing a Tecan Infinite F200 plate reader.
HT-29 cell differentiation
HT-29 cells had been cultured in RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine, 100 U ml–1 penicillin, 100 μg ml–1 streptomycin and a couple of g l–1 glucose for 21 days, as described33. The cell medium was modified each different day till the cells reached confluence. Thereafter, the medium was modified each day for 21 days. The differentiated cells had been then uncovered to GO (30 μg ml–1) for twenty-four h. Then, the cells had been washed, harvested utilizing trypsin–EDTA (0.25%) and glued in 2.50% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The samples had been then processed for TEM evaluation, as described within the ‘Ultrastructural evaluation’ part.
Zebrafish had been housed and experiments had been carried out in compliance with nationwide moral pointers, and the current examine was authorised by the regional committee for animal experiments in Stockholm (moral allow no. 14049-2019). Zebrafish embryos carrying a degree mutation in ahr2 (ahr2hu3335) had been generated on the Wellcome Sanger Institute and supplied by the European Zebrafish Useful resource Heart at Karlsruhe Institute of Know-how. Offspring had been raised to maturity and genotyped for the ahr2hu3335 level mutation with DNA remoted from fin clips34. DNA was extracted utilizing the QIAamp DNA Mini Equipment (QIAGEN) and amplified with the purpose mutation detection primers (ahr2-mut-F, TATTGCTAGGCAGAGAGCAC; ahr2-mut-R, GATGTCTTCTGTGATGATTTCG) utilizing the DreamTaq Inexperienced PCR Grasp Combine (ThermoFisher). The PCR product was purified with ExoSAP-IT Specific reagent (Utilized Biosystems), and loaded on the ABI 3730 PRISM DNA analyser (Utilized Biosystems) for DNA sequencing. Zebrafish decided to be WT (ahr2+/+), heterozygous (ahr2+/−) or homozygous (ahr2−/−) for the purpose mutation in ahr2 had been used for additional experiments (grownup zebrafish, 4.5 months previous; larvae, 5 dpf).
Grownup zebrafish publicity
Grownup zebrafish of various genotypes (WT, ahr2+/− and ahr2−/−) had been constantly uncovered to GO (50 µg l–1 or 500 µg l–1) for seven days. For WT, six feminine and 6 male fish had been included, whereas for the opposite genotypes, three feminine and three male fish had been included. The zebrafish had been housed collectively earlier than the genotyping (see above) and had been then housed in separate fish tanks for one week earlier than the exposures to GO. Through the seven-day publicity, the fish had been fed as soon as per day within the morning with an roughly equal quantity for every tank. The fish water was refreshed 1 h after the feeding and GO was added to the publicity teams. At day 7, the fish had been sacrificed with tricaine, and the intestines had been dissected beneath a stereomicroscope and glued in 2.5% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 and saved at 4 °C for TEM evaluation (WT), or in 4% formaldehyde for histopathological examination (WT) or saved at −80 °C for subsequent 16S rRNA gene sequencing (WT, ahr2+/−). The ahr2+/− zebrafish had been used for the evaluation of the intestine microbiome composition because the survival of some ahr2−/− fish within the high-dose GO publicity group was compromised.
After fixation in 4% formaldehyde for at the least 24 h, the intestines had been dehydrated in ethanol and embedded in paraffin utilizing an embedding station (Tissue-Tek, Sakura Finetek). The paraffin-embedded tissues had been then lower utilizing a microtome (Microm HM 360, Marshall Scientific). The thickness of every slice was 5 µm. The slices had been deparaffinized following the steps of xylene for five min, xylene for five min, 100% ethanol for 3 min, 95% ethanol for 3 min and distilled water for 3 min. Sections had been stained with haematoxylin and eosin for common morphological examination, and Alcian blue and periodic acid–Schiff reagent (ThermoScientific) for goblet cell identification utilizing a Zeiss Axioplan microscope geared up with an Olympus SC30 digital digicam. The numbers of goblet cells per villus are offered as common outcomes of six slices per situation.
TEM evaluation35 of the GI tract of management and uncovered animals was carried out on 5 dpf larvae and dissected intestines of grownup zebrafish. Following the first fixation, samples had been rinsed with 0.1 M phosphate buffer adopted by post-fixation in 2% osmium tetroxide in 0.1 M phosphate buffer at pH 7.4 at 4 °C for two h. The samples had been then ethanol dehydrated stepwise adopted by stepwise acetone/LX-112 infiltration and at last embedded in LX-112. Semi- and ultrathin sections had been ready on a Leica EM UC7 ultramicrotome. The ultrathin sections had been then contrasted with uranyl acetate adopted by Reynolds lead citrate and examined utilizing a Hitachi HT7700 transmission electron microscope working at 100 kV. Digital photos had been acquired utilizing a 2k × 2k Veleta charge-coupled machine digicam.
16S rRNA gene sequencing
Complete DNA of the zebrafish intestine samples was extracted utilizing the QIAamp DNA Mini Equipment (QIAGEN). The amplicon PCR was then carried out with bacterial common primers to focus on the V3 and V4 areas of the 16S rRNA gene utilizing the ahead primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCA and the reverse primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTA CHVGGGTATCTAATCC. The KAPA HiFi HotStart ReadyMix (KAPA Biosystems) was used for the PCR reactions. Index PCR was carried out with the Nextera XT Index Equipment (Illumina), adopted by cleanup with AMPure XP beads. The concentrations of the DNA libraries had been quantified by an Agilent 2100 Bioanalyser (Agilent Applied sciences). DNA libraries had been then sequenced on the Illumina MiSeq system to generate uncooked paired-end reads (2 × 150 bp).
Gene sequencing evaluation
Primers had been faraway from sequences utilizing cutadapt (model 2.9) and sequences had been high quality checked by FastQC (model 0.11.9) and mixed utilizing multiqc (model 1.9.dev0). ASVs had been inferred from the bacterial 16S rRNA gene sequences had been utilizing the DADA2 bundle36 (model 1.14.1). Ahead and reverse reads had been truncated after 282 and 222 bp, respectively, and additional filtered utilizing the perform filterAndTrim with default choices, with the exception that the utmost anticipated error price was set at 2. The remainder of the features within the DADA2 pipeline had been carried out with default choices, with the exception that earlier than pattern inference, that’s, eradicating sequencing errors, all of the samples had been pooled somewhat than dealt with pattern sensible (default). The ASVs of 16S rRNA gene sequences had been assigned to taxa utilizing the SILVA taxonomic coaching information formatted for DADA2 (model 138)37. Earlier than the normalization step, non-bacterial sequences had been eliminated. ASVs had been then normalized utilizing cumulative sum scaling38. The steps within the evaluation of the info from preprocessing to additional downstream analyses had been executed within the R setting (3.6.2, R Core Group, 2019).
Earlier than unsupervised and supervised analyses of the preprocessed and normalized information, ASVs occurring solely in a single pattern had been eliminated. To analyze the general variation within the intestine microbiota of WT and AhR-deficient zebrafish, principal coordinate evaluation was carried out utilizing the perform cmdscale within the R bundle vegan39. Supervised analyses had been, in flip, used to review the impact of GO publicity on the intestine microbiota of WT and ahr2+/− animals, making an allowance for the gender of the animals. Permutational multivariate evaluation of variance (PERMANOVA) and distance-based redundancy evaluation (dbRDA) had been carried out utilizing the features adonis and dbrda, respectively, within the R bundle vegan. Within the PERMANOVA and dbRDA analyses, publicity, gender and genotype had been utilized as categorical variables to oversee the microbiota composition. Between-sample Bray–Curtis distances had been utilized in principal coordinate evaluation, PERMANOVA and dbRDA. Statistical significances had been based mostly on 9,999 random permutations. To find out which ASVs had been in another way considerable in exposures and genotypes, the perform fitFeatureModel was utilized in R bundle metagenomeSeq (retrieved from https://cbcb.umd.edu/software program/metagenomeSeq).
GF zebrafish derivation
The technology of GF zebrafish adopted beforehand established protocols40. In short, 2 hpf embryos had been transferred to Petri dishes with sterile E3 medium, supplemented with ampicillin (100 μg ml–1), kanamycin (5 μg ml–1) and amphotericin B (250 ng ml–1), and incubated at 28 °C. At 50% epiboly as much as the defend stage (6 hpf), the embryos had been floor disinfected with 0.1% polyvinylpyrrolidone-iodine for precisely 2 min, adopted by 0.003% sterile bleach immersion for 18 min. The embryos had been then rinsed with sterile E3 medium, transferred to flasks and incubated at 28 °C. The viability was monitored, and the sterile medium was refreshed every day. At day 4, the hatched embryos had been used for sterility validation. Gnotobiotic zebrafish had been validated by way of two completely different approaches: bacterial development on Luria broth plates and DNA amplification by bacterial common primers. To this finish, ten embryos had been randomly chosen from the tradition flasks. For the primary strategy, the embryos had been washed and homogenized with 200 μl sterile medium. Then, 100 μl homogenate was unfold on the Luria broth plate and incubated at 37 °C in a single day. The bacterial colony formation was checked on the following day. For the second strategy, DNA of the collected embryos was extracted utilizing the QIAamp DNA Mini Equipment (QIAGEN). DNA was amplified with bacterial common primers (515F/806R) utilizing the DreamTaq Inexperienced PCR Grasp Combine (ThermoFisher). The PCR product was resolved by electrophoresis, and Midori Inexperienced Direct (NIPPON Genetics Europe) was used for the visualization of the DNA. The gel picture was captured utilizing a Gel Doc EZ System (Bio-Rad).
Zebrafish larvae publicity
WT and ahr2−/− grownup zebrafish had been maintained at 28.0 ± 0.5 °C on a 14 h:10 h mild/darkish cycle within the fish breeding circulatory system on the zebrafish core facility at Karolinska Institutet. Two pairs of male/feminine fish had been positioned in a single mating tank with a divider sooner or later earlier than spawning. Spawning was triggered by eradicating the divider within the morning. Embryos had been collected after 2 h, washed after which transferred to the E3 medium in a Petri dish. Wholesome and fertilized embryos on the similar developmental phases had been chosen and raised as much as 5 dpf. Zebrafish larvae at 5 dpf (CV WT, GF WT, CV ahr2−/− and GF ahr2−/−) had been uncovered to GO or BA or GO+BA for twenty-four h. Every remedy was carried out in three replicates, and every replicate pattern contained ten larvae. FICZ (200 nM) was used as a optimistic management for cyp1a induction. After the publicity, the samples had been fastened in 2.5% glutaraldehyde for TEM evaluation or saved at −80 °C for RT-qPCR, as detailed above.
Raman confocal evaluation
The presence of GO within the GI tract of zebrafish larvae was monitored by Raman confocal evaluation11. Briefly, larvae uncovered to GO for twenty-four h had been washed, anaesthetized in 0.01% tricaine answer and positioned in 1% low-melt agarose on glass slides. Samples had been then dried on a plate heater at 50 °C. Raman evaluation was carried out utilizing a confocal Raman microscope (WITec alpha300 system) with a laser of 532 nm wavelength set at an integration time of 0.5 s and ×60 magnification. The scan space for every pattern was adjusted to 50 × 50 µm2. The spectra proven symbolize the common of 10,000 spectra recorded throughout the entire space scan. GO could possibly be detected on the idea of its attribute Raman signature, that’s, the D band (1,354 cm−1), G band (1,582 cm−1) and 2D band (2,690 cm−1).
RNA extraction was carried out on 5 dpf larvae (WT versus ahr−/−) uncovered to GO+BA or not utilizing the RNeasy Mini Equipment (QIAGEN). RNA focus was quantified by a NanoDrop spectrophotometer (ThermoFisher). Complete RNA (500 ng) was reverse transcribed utilizing the iScript Superior cDNA Synthesis Equipment (Bio-Rad). The transcription of goal genes was quantified utilizing a QuantStudio 5 Actual-Time PCR System (Utilized Biosystems). The response mixtures had been formulated utilizing Maxima SYBR Inexperienced/ROX qPCR Grasp Combine (ThermoScientific). Thermal biking situations had been as follows: 95 °C for 10 min, 40 cycles of three-step amplification of 15 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C. Primers (Supplementary Desk 3) had been bought from Sigma-Aldrich. The transcription degree of every goal gene was normalized to rpl13. The relative mRNA expression degree was calculated relative to regulate utilizing the two−ΔΔCt technique.
Tg(cyp1a:GFP) reporter pressure
Transgenic Tg(cyp1a:GFP) zebrafish41 had been supplied by the China Zebrafish Useful resource Heart. GF zebrafish larvae had been generated as described above and uncovered at 5 dpf to GO (30 µg ml–1), pink fluorescent resorufin butyrate (Sigma-Aldrich) (5 µM) and a mix of GO and resorufin butyrate for twenty-four h. FICZ (Sigma-Aldrich) (200 nM) was used as a optimistic management. After publicity, the larvae had been washed, anaesthetized in 0.01% tricaine and positioned in 1% low-melt agarose for evaluation by confocal microscopy (Zeiss LSM880, ZEISS). The intense-field photos had been acquired with the transmitted mild detector (T-PMT). The inexperienced and pink fluorescence photos had been captured beneath 488 and 561 nm to visualise cyp1a and butyrate, respectively. The pictures had been analysed in ZEN 3.0 software program, blue version (ZEISS). The two.5D view instrument (ZEN) was used to generate pseudo-three-dimensional photos and the highest-intensity values are represented by the best extension within the z axis.
Tg(lck:GFP) reporter pressure
Transgenic Tg(lck:GFP) zebrafish42 had been obtained by way of the European Zebrafish Useful resource Heart. CV and GF larvae had been uncovered at 5 dpf to a mix of GO (30 µg ml–1) and butyrate (2.5 mM) for twenty-four h. GO and BA had been pre-incubated for 1 h earlier than the publicity. After the publicity, the larvae had been washed, anaesthetized and positioned for evaluation by confocal microscopy (Zeiss LSM880, ZEISS), as described above. The numbers of lck-positive cells homing to the intestine had been manually quantified based mostly on seven fish per group. Resorufin butyrate (5 µM) was additionally utilized to raised visualize the interactions between GO, butyrate and lck-positive cells beneath GF situations. The z-stack evaluation was carried out with the interval of 1 µm of every slice, and z projections had been made with Fiji (ImageJ)43.
Zebrafish dissociation and cell sorting
GF WT (AB) zebrafish larvae had been uncovered at 5 dpf to the mixture of GO (30 µg ml–1) and BA (2.5 mM) for twenty-four h. GO and BA had been pre-incubated for 1 h earlier than the publicity. Twenty larvae had been used as one replicate, and 4 replicates, that’s, eighty larvae in complete, had been used for every situation. After the publicity, zebrafish larvae had been dissociated for single-cell suspensions following the revealed protocol44. Briefly, zebrafish larvae had been euthanized with 0.01% tricaine for five min, collected in a 1.5 ml tube and washed thrice with phosphate-buffered saline. The dissociation was initiated by including 500 μl of pre-warmed enzyme combine containing 460 μl of 0.25% trypsin–EDTA (Gibco) and 40 μl of collagenase (Sigma-Aldrich) (100 mg ml–1), adopted by mechanical dissociation utilizing P1000 after which P200 pipette tips about a warmth block at 30 °C till tissues had been now not seen (about 10 min). The dissociation was then stopped by including 800 μl DMEM supplemented with 10% FBS. The cell pellets had been collected by centrifugation at 1,000 rpm for five min at room temperature, adopted by washing with phosphate-buffered saline. The cells had been then resuspended in 0.5 ml DMEM + 10% FBS. 4 replicates from every situation had been pooled collectively at this step and filtered by way of a 40 μm nylon mesh, with an extra washing step with DMEM + 10% FBS. The cell suspension was then stained for 10 min at room temperature with the fluorescent DNA dye DRAQ7 (Invitrogen) (3 μM) to permit for the exclusion of non-viable cells through the use of fluorescence-activated cell sorting (BD FACSAria III, BD Biosciences) working with FCS Specific software program model 7.0 (DeNovo Software program). DRAQ7– cells of every pattern had been sorted into tubes containing DMEM + 10% FBS and had been instantly positioned on ice and proceeded additional for scRNA-seq evaluation, as described under. Along with the sorting of cells from WT embryos, cells had been additionally sorted from GF transgenic Tg(lck:GFP) zebrafish to complement lck+ cells for scRNA-seq. Fifty larvae had been used as one replicate, and 4 replicates, that’s, 200 larvae in complete, had been used for every situation. The publicity, and the procedures for single-cell dissociation and DRAQ7 staining, had been the identical as for the WT zebrafish. Nonetheless, the gating technique was based mostly on ahead scatter, DRAQ7 and GFP. DRAQ7−GFP+ cells had been sorted into tubes containing DMEM + 10% FBS and had been instantly positioned on ice, and processed for scRNA-seq evaluation.
Single-cell RNA sequencing
The samples had been loaded on a 10x GemCode Single-Cell Instrument (10x Genomics) to generate single-cell gel beads in emulsion (GEMs), and libraries had been constructed utilizing the Chromium Subsequent GEM Single Cell 3’ GEM, Library & Gel Bead Equipment v3.1 (10x Genomics). Briefly, GEMs had been generated by combining barcoded Single Cell 3’ v3.1 Gel Beads, a Grasp Combine containing cells and partitioning oil onto Chromium Subsequent GEM Chip G. Following GEM technology, the gel bead was dissolved, primers containing an Illumina TruSeq Learn 1, 10x Barcode, distinctive molecular identifier and poly(dT) sequence had been launched, and the cells had been lysed. The barcoded, full-length cDNA was synthesized, purified and amplified by PCR for library building. Twin-indexed libraries containing the P5 and P7 primers utilized in Illumina amplification had been ready for an estimated 5,000 nuclei per pattern. Paired-end, twin indexing sequencing of libraries was carried out on a NovaSeq 6000 sequencing system (Illumina). Cell Ranger 6.0.1 (10x Genomics) pipelines (cellranger mkfastq and cellranger rely) had been used to transform Illumina Base name information to FASQT format, align sequencing reads to the zebrafish reference genome GRCz11 and generate feature-barcode matrices. The generated feature-barcode matrices had been used for the next evaluation.
Information evaluation of 10x Genomics information
The evaluation of the 10x Genomics information was carried out utilizing the Seurat toolkit (model 4.0.6) (out there at https://satijalab.org/seurat/index.html) within the R setting (RStudio, model 4.2.0). A typical preprocessing workflow was utilized, together with QC metrics, information normalization and scaling, and the detection of extremely variable options. Particularly, Chromium Single Cell 3’ samples with distinctive function counts over 6,000 or lower than 200 had been filtered out. As well as, the share of mitochondrial content material was set to be lower than 10%. After QC, 3,115 single cells from the WT management pattern and three,012 single cells from the WT GO+BA pattern (experiment 1) and a couple of,312 single cells from the Tg(lck:GFP) management pattern and a couple of,669 single cells from the Tg(lck:GFP) GO+BA pattern (experiment 2) had been used for the downstream evaluation. The uncooked counts that handed the QC had been centred by a scale issue of 10,000 and log remodeled. The extremely variable options had been detected utilizing the ‘FindVariableFeatures’ command in Seurat by immediately modelling the imply–variance relationship inherent within the single-cell information and a couple of,000 options per dataset had been returned45. A linear transformation (scaling) was utilized to the info earlier than the principal part evaluation. Solely the beforehand decided extremely variable options had been used because the enter for the calculation of principal elements utilizing the ‘RunPCA’ command in Seurat. The ‘JackStrawPlot’ and ‘ElbowPlot’ instructions had been utilized to visualise the rating of the principal elements. Important principal elements displaying sturdy enrichment of options with low p values had been chosen for the next clustering evaluation46. Particularly, 20 and 15 principal elements had been recognized for the primary and second RNA-seq experiment, respectively. The t-stochastic neighbour embedding transformation47 was achieved by the ‘RunTSNE’ command in Seurat. The optimistic marker genes in every cluster in contrast with all of the remaining cells had been recognized utilizing the ‘FindAllMarkers’ command. An recognized function requires to be detected at a minimal proportion of 0.25 in every of the cluster of cells and differentially expressed (on common) with a Log2FC threshold of 0.25 between the clusters. The cell clusters had been annotated in response to the Zebrafish Info Community (https://zfin.org/) and on the idea of present literature25,48,49. The visualization of the marker expression was demonstrated by warmth maps, violin plots, function plots and dot plots utilizing the ‘DoHeatmap’, ‘VlnPlot’, ‘FeaturePlot’ and ‘DotPlot’ instructions, respectively. The scRNA-seq integration evaluation was additional carried out to establish the cell sorts which might be current in each datasets and to search out cell-type-specific responses to the stimulation50. The normalization and identification of variable options for every dataset had been independently carried out as described above. The options that had been repeated variables throughout the datasets had been chosen for integration utilizing the ‘SelectIntegrationFeatures’ command in Seurat, adopted by ‘FindIntegrationAnchors’ and ‘IntegrateData’ instructions to create an built-in information assay. The usual workflow described above, together with scaling information, principal part evaluation, clustering and t-stochastic neighbour embedding transformation was additionally carried out on the built-in information. The ‘break up.by’ argument was used to visualise the 2 situations facet by facet, and the ‘subset’ argument was used to plot the info in a selected cluster. The transcriptomics information from each analyses are deposited in ArrayExpress at EMBL-EBI.
Experiments had been carried out at the least thrice, every in triplicate for every situation. Statistical variations had been analysed utilizing the Pupil’s t-test (GraphPad Prism model 8.2.0). The info offered are imply values ± customary deviation (s.d.). The variations between teams or therapies had been thought-about vital when p < 0.05. The evaluation of the 16S rRNA information and scRNA-seq information is described above.
Additional info on analysis design is on the market within the Nature Portfolio Reporting Abstract linked to this text.