Mardiyyah/TAPT_data_V2_split · Datasets at Hugging Face

PMCID stringclasses 24 values	Title stringclasses 24 values	Sentences stringlengths 2 40.7k
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	This shift toward progenitor states underpins CD-associated malabsorption, beyond reduction in intestinal surface area.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	In contrast to the term ‘villus atrophy’, we observe that the CD epithelium is hyperproliferative, and so the loss of villus structures requires additional explanation.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Spatial transcriptomics data indicate specific regions responsible for WNT signaling, and we hypothesize that CD inflammation drives morphogen signaling shifts causing mucosal remodeling.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	These morphogen responses may not be CD specific, and may underpin histological similarities seen with mimics such as environmental enteropathy, monogenic enteropathies and olmesartan enteropathy.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Integrating single-cell and spatial transcriptomics data, we have dissected the molecular and cellular basis of the histological changes in CD, including villus epithelial changes, crypt hyperplasia and intraepithelial lymphocytosis.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	This cellular, spatial transcriptomics description builds on the Marsh–Oberhuber histological description, with complex, highly localized, mucosal cell communities, including focal lymphoid organization where specific cell types, including gluten-specific TFH-like CD4 T cells, B cells and Treg cells, are co-located.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Overall, our study of the mucosal cellular and spatial landscape in CD provides a detailed foundation from which to explore potential therapeutic targets, and highlights the need to explore the clinical implications of the prolonged epithelial–immune scar in TCD.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Study participants with CD, and HCs, were identified via Oxford University Hospitals NHS Trust CD clinic and endoscopy service (Oxford, UK).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Blood and intestinal biopsy samples were taken at endoscopy with informed consent under the Oxford Gastrointestinal Illnesses Biobank study (REC: 21/YH/0206).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Study participant demographics and study inclusion/exclusion criteria are summarized in Supplementary Table 1.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Participants were not compensated financially.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Peripheral blood mononuclear cells were extracted from whole blood or leukocyte cones via density gradient centrifugation.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Briefly, peripheral blood was diluted at a 1:1 ratio with Dulbecco’s phosphate buffered saline without calcium or magnesium (PBS) and layered over Lymphoprep (Axis-Shield) before centrifugation (973g for 30 min at 20 °C without brake).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	The mononuclear layer was retrieved and washed twice in PBS or R10 culture media (RPMI-1640 (Sigma-Aldrich), 10% FCS, 1% penicillin/streptomycin, 1% l-glutamine).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Viable mononuclear cells stained with Trypan blue were counted manually by microscopy using a hemocytometer before downstream applications.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	If remnant red blood cells were present, they were lysed with ammonium–chloride–potassium solution for 2–3 min, then washed again in R10.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Samples were cryopreserved in freezing medium (90% FCS (Sigma-Aldrich), 10% dimethylsulfoxide (Sigma-Aldrich)).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	When needed, samples were thawed rapidly in a water bath (37 °C), then washed twice in R10 before downstream use.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Intestinal biopsy samples were collected at endoscopy from duodenum.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Biopsy samples for intestinal lymphocyte extraction were immediately placed in sterile R10 medium (as above) or MACS Tissue Storage Solution (Miltenyi Biotec) on ice for transportation, before cryopreservation in CryoStor Cs10 (STEMCELL Technologies).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	This approach preserves immune cell viability and surface marker expression.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	When required, samples were rapidly thawed in a 37 °C water bath and washed in 20 ml R10 before tissue dissociation.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	For immune cell isolation from duodenal biopsy samples for scRNA-seq (10x Genomics, dataset 1), biopsy samples were incubated in R10 medium with 1 mg ml Collagenase D (Roche) and 100 mg ml DNase (Thermo Fisher Scientific) for 1 h in a shaking incubator at 37 °C.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Biopsy samples were then dissociated by vigorous agitation using a GentleMACS Dissociator (Miltenyi Biotec), then strained through a 70-μm filter.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	The mononuclear cells were isolated on a discontinuous 70% and 35% Percoll gradient (GE Healthcare) by centrifugation at 700g for 20 min without brake.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Mononuclear cells were collected from the interface and washed in R10.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Cells were washed with R10 medium before antibody staining and downstream applications.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	The complete protocol with all steps is available at https://www.protocols.io/view/freezing-and-processing-intestinal-biopsies-for-th-dm6gp8745lzp/v1/ (Oxford HCA, 2019).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	The method for EC isolation from duodenal biopsy samples for scRNA-seq (10x Genomics, dataset 1) was adapted from ref. .
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Biopsy samples were washed in wash medium (HPGA, 1 mM EDTA, 1 mM dithiothreitol), then incubated in chelation medium (HPGA, 1 mM EDTA) at 37 °C for 40 min with agitation.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	The supernatant, which was removed and replaced every 10 min and contained epithelial crypts, was digested into a single-cell suspension by dissociation in a shaking incubator with TrypLE Express and DNase (50 µg ml) for 60 min at 37 °C.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	The epithelial single-cell suspension was washed with PBS and passed through a 30-µm filter.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Cell counts and viability were confirmed with a manual hemocytometer before further processing.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	The complete protocol with all steps is available at https://www.protocols.io/view/isolation-of-cells-from-the-epithelial-layer-of-fr-e6nvw9e6dgmk/v1/ (Oxford HCA, 2019).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	For immune cell isolation from duodenal biopsy samples for scRNA-seq and proteomics (BD Rhapsody, dataset 2), samples were thawed, then diluted with warm X-VIVO (Lonza) + 1% AB serum (Sigma-Aldrich).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Biopsy samples underwent enzymatic and mechanical digestion using 0.042 mg ml Liberase TL (Roche) and 1 mg ml DNAse I (Thermo Fisher).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Samples were placed horizontally in a shaking incubator for 20 min at 37 °C and homogenized using a gentleMACS Dissociator (Miltenyi Biotech).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Dissociated cells were passed through a 70-μm strainer.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Lymphocytes were enriched by Percoll density centrifugation as above.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Duodenal biopsy samples for flow cytometry were processed as for scRNA-seq of immune cells (see above).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Cells were washed with R10 medium before antibody staining and downstream applications.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Duodenal biopsy samples for fluorescence-activated cell sorting (FACS) of IELs for RNA-seq and TCR repertoire sequencing were placed in 10 ml HBSS with 1 mM EDTA and 1 mM dithiothreitol (both Sigma-Aldrich) and placed in a shaking incubator (200 rpm, 37 °C) for 15 min.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	IELs were strained through a 70-μm filter and washed two to three times with R10 before downstream applications.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	For spatial transcriptomics, single intestinal biopsy samples were embedded in OCT cryo-embedding matrix (Thermo Fisher Scientific) then frozen in isopentane (Sigma-Aldrich) suspended over liquid nitrogen or dry ice, and stored at −80 °C until use.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	For surface marker staining, cells were stained in 50 ml of FACS buffer (PBS + 1 mM EDTA + 0.05% BSA) for 30 min at 4 °C.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Surface antibodies and clones used are listed in Supplementary Table 3.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Antibodies were purchased from BioLegend, BD Biosciences, Miltenyi Biotec or Thermo Fisher Scientific.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	After staining, cells were stored at 4 °C protected from light until data acquisition.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Flow cytometry data were acquired on a BD LSR II flow cytometer (BD Biosciences).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	FACS samples were surface stained as above, with Sytox Green (Thermo Fisher Scientific) used as a viability dye.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	FACS was performed on an Aria III (BD Biosciences; 70-mm nozzle).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	For sorting by FACS for scRNA-seq of intestinal immune populations, cells were stained with EpCam-PE, CD27-BV421 and CD45-APC-Cy7.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Live CD45 or CD27 cells were sorted to include all mucosal immune cell populations, including long-lived CD27 plasma cells, which can downregulate surface CD45 expression.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	For sorting by FACS for scRNA-seq of intestinal epithelial populations, cells were stained with EpCAM-PE and CD45-AF700, with live EpCAM cells sorted.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	For sorting by FACS for bulk RNA-seq or TCR-seq of CD8 IEL populations, cells were stained with CD45-BV785, CD3-BV711, αβTCR-APC, γδTCR-PE, CD4-BV650 and CD8a-AF700, with live CD45CD3αβTCRCD8CD4 cells sorted.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Libraries were generated using 10x Genomics Chromium Single Cell V(D)J Reagents Kits (v1 Chemistry) per the manufacturer’s instructions.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Sorted cells suspended in PBS (plus 0.04% BSA) at a concentration of 1,000 cells per microliter were loaded into one lane of a Chromium controller.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Library quality and quantity were assessed using a TapeStation (Agilent) and Qubit Fluorometer (Thermo Fisher Scientific).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Libraries were sequenced on an Illumina HiSeq 4000 following the manufacturer’s instructions.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Library generation and sequencing were performed at the Sanger Institute, Cambridge.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Sorted CD45 cells were stained with a cocktail of 79 oligonucleotide-conjugated AbSeq antibodies (BD Biosciences, for 45 min at 4 °C.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Cells were then washed to remove residual unbound AbSeq antibodies and loaded onto three BD Rhapsody cartridges (BD Biosciences) for single-cell capture.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	AbSeq antibodies used in this study are listed in Supplementary Table 2.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Single-cell capture and cDNA library preparation were performed using the BD Rhapsody Express Single-Cell Analysis System (BD Biosciences), according to the manufacturer’s instructions.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Briefly, cDNA was amplified—ten cycles for resting cells and nine cycles for in vitro-stimulated cells—using the Human Immune Response Primer Panel (BD Biosciences; Supplementary Table 6), containing 399 primer pairs and a supplementary panel of 105 primer pairs (BD Biosciences; Supplementary Table 3).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	The resulting PCR1 products were purified using AMPure XP magnetic beads (Beckman Coulter), and the respective mRNA and AbSeq/Sample Tag products were separated based on size selection, using different bead ratios (0.7× and 1.2×, respectively).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	The purified mRNA and Sample Tag PCR1 products were further amplified (ten cycles), and the resulting PCR2 products purified by size selection (1× and 1.2× for the mRNA and Sample Tag libraries, respectively).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	The concentration, size and integrity of the resulting PCR products were assessed using both Qubit (High-Sensitivity dsDNA Kit; Thermo Fisher) and the Agilent 4200 TapeStation system (High Sensitivity D1000 ScreenTape; Agilent).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	The final products were normalized to 2.5 ng μl (mRNA), 0.5 ng μl (Sample Tag) and 0.275 ng μl (AbSeq) and underwent a final round of amplification (six cycles for mRNA and eight cycles for Sample Tag and AbSeq) using indexes for Illumina sequencing to prepare the final libraries.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Final libraries were quantified using Qubit and Agilent TapeStation and pooled (~60%/38%/2% mRNA/AbSeq/Sample Tag ratios, respectively) to achieve a final concentration of 5 nM. Final pooled libraries were spiked with 10% PhiX control DNA to increase sequence complexity and sequenced (75 base pairs (bp), paired-end) on a HiSeq 4000 sequencer (Illumina).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Cryopreserved, OCT-embedded duodenal biopsy samples were stored at −80 °C until use.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Before performing the full protocol, a tissue permeabilization optimization was performed (10x Genomics, Visium Spatial Tissue Optimization), which identified 11 min as the optimum permeabilization time.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Samples were processed for spatial transcriptomics per the manufacturer’s instructions (10x Genomics, Visium Spatial), with 2 × 10-μm sections cut on a pre-cooled cryostat for each sample onto two 6.5 × 6.5-mm capture areas, each with approximately 5,000 oligonucleotide-barcoded 55-μm-diameter spots.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Slides were fixed, H&E stained and imaged on a Leica DMI8 Widefield microscope at a magnification of ×40.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Tissue was permeabilized per instructions for 11 min, followed by reverse transcription and second-strand synthesis performed on the slide.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	cDNA quantification was performed using qPCR using KAPA SYBR FAST-qPCR kit (KAPA Biosystems) on a CFX96 Thermal Cycler instrument (Bio-Rad).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Following library construction per instructions, the spatial transcriptomics libraries were quantified and pooled at a concentration of 4 nM with a sample ratio corresponding to the approximate surface area of tissue coverage obtained from the H&E imaging.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Pooled libraries were sequenced on a NextSeq (Illumina) using a 150-bp paired-end dual-indexed setup (High output, v2.5, Illumina) loaded at a concentration of 1.8 pM, and sequenced to a manufacturer-recommended depth of a minimum of 50,000 reads per tissue-covered spot.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Cryopreserved, OCT-embedded duodenal biopsy samples were stored at −80 °C until use.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Spatial transcriptomics was performed using the Visium CytAssist (10x Genomics) workflow for fresh-frozen tissue according to the manufacturer’s instructions.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Ten-micron sections were cut on a cryostat pre-cooled to −20 °C and placed on 11 × 11-mm areas (four sections per area) on SuperFrost Plus slides (Thermo Fisher).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Sections were fixed, H&E stained and imaged on an Axioscan Z1 slide scanner (Zeiss) at a magnification of ×20.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Sections were de-stained and subjected to on-slide probe hybridization and ligation followed by probe transfer onto Visium CytAssist Spatial Gene Expression slides, each containing an 11 × 11-mm capture area covered by approximately 14,000 55-µm-diameter oligonucleotide-barcoded spots.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Probes were extended and cDNA quantified by qPCR using a KAPA SYBR FAST-qPCR kit (KAPA Biosystems) on a CFX96 Thermal Cycler instrument (Bio-Rad), followed by off-slide library construction per the instructions.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Libraries were quantified and pooled at a concentration of 2 nM with a sample ratio corresponding to the approximate surface area of tissue coverage obtained from the H&E imaging.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Pooled libraries were sequenced on a NextSeq 500 instrument (Illumina) using a 150-bp paired-end dual-indexed setup (High output, v2.5, Illumina) at a manufacturer-recommended depth of a minimum of 50,000 reads per tissue-covered spot.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	A TRIzol nucleic acid extraction method was used to extract RNA from low numbers of sorted lymphocytes, as previously described, except that phase-lock gel tubes were replaced with standard 1.5 ml microcentrifuge tubes.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Briefly, after sorting, cells were centrifuged (500g, 5 min), resuspended in 1 ml TRIzol, then frozen at −80 °C until RNA extraction.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	For RNA extraction, samples were thawed, mixed with 200 μl chloroform and centrifuged (14,000g, 5 min).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	A total of 500 μl of the aqueous phase was taken and RNA was extracted using the Agencourt RNAdvance Tissue Isolation kit.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	RNA concentration and purity were assessed on a 2100 Bioanalyzer instrument (Agilent).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Bulk RNA-seq was performed using the Smart-seq2 protocol at the Oxford Genomics Centre (University of Oxford).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Around 10 ng RNA was used as a template from each sample for library generation.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Barcoded samples were pooled, and External RNA Controls Consortium RNA (1:100,000 dilution) was added before 75-bp paired-end sequencing on an Illumina HiSeq 4000 instrument.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Bulk TCR repertoire sequencing was performed using the amplicon-rescued multiplex-PCR method (iRepertoire).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	This method performs an initial first-round RT–PCR with TCR V and C gene-specific primers for the relevant TCR chain, followed by further amplification steps with universal primers for the exponential phase of amplification.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	This method is designed to provide quantitative, deep sequencing of the TCR repertoire, with minimal bias.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Library generation was performed following the manufacturer’s instructions, except for using 96-well plates.
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	The quality, size distribution, concentration and presence of contaminating primer dimers of the final product was assessed using agarose gel electrophoresis, a spectral photometer (Nanodrop, Thermo Fisher Scientific), and the Bioanalyser DNA 1000 assay using a 2100 Bioanalyzer instrument (Agilent).
PMC12133578	Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.	Libraries were quantified using the KAPA Library Quantification Kit (Roche) on a CFX96 Thermal Cycler instrument (Bio-Rad) before equimolar pooling.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

This shift toward progenitor states underpins CD-associated malabsorption, beyond reduction in intestinal surface area.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

In contrast to the term ‘villus atrophy’, we observe that the CD epithelium is hyperproliferative, and so the loss of villus structures requires additional explanation.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Spatial transcriptomics data indicate specific regions responsible for WNT signaling, and we hypothesize that CD inflammation drives morphogen signaling shifts causing mucosal remodeling.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

These morphogen responses may not be CD specific, and may underpin histological similarities seen with mimics such as environmental enteropathy, monogenic enteropathies and olmesartan enteropathy.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Integrating single-cell and spatial transcriptomics data, we have dissected the molecular and cellular basis of the histological changes in CD, including villus epithelial changes, crypt hyperplasia and intraepithelial lymphocytosis.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

This cellular, spatial transcriptomics description builds on the Marsh–Oberhuber histological description, with complex, highly localized, mucosal cell communities, including focal lymphoid organization where specific cell types, including gluten-specific TFH-like CD4 T cells, B cells and Treg cells, are co-located.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Overall, our study of the mucosal cellular and spatial landscape in CD provides a detailed foundation from which to explore potential therapeutic targets, and highlights the need to explore the clinical implications of the prolonged epithelial–immune scar in TCD.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Study participants with CD, and HCs, were identified via Oxford University Hospitals NHS Trust CD clinic and endoscopy service (Oxford, UK).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Blood and intestinal biopsy samples were taken at endoscopy with informed consent under the Oxford Gastrointestinal Illnesses Biobank study (REC: 21/YH/0206).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Study participant demographics and study inclusion/exclusion criteria are summarized in Supplementary Table 1.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Participants were not compensated financially.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Peripheral blood mononuclear cells were extracted from whole blood or leukocyte cones via density gradient centrifugation.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Briefly, peripheral blood was diluted at a 1:1 ratio with Dulbecco’s phosphate buffered saline without calcium or magnesium (PBS) and layered over Lymphoprep (Axis-Shield) before centrifugation (973g for 30 min at 20 °C without brake).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

The mononuclear layer was retrieved and washed twice in PBS or R10 culture media (RPMI-1640 (Sigma-Aldrich), 10% FCS, 1% penicillin/streptomycin, 1% l-glutamine).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Viable mononuclear cells stained with Trypan blue were counted manually by microscopy using a hemocytometer before downstream applications.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

If remnant red blood cells were present, they were lysed with ammonium–chloride–potassium solution for 2–3 min, then washed again in R10.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Samples were cryopreserved in freezing medium (90% FCS (Sigma-Aldrich), 10% dimethylsulfoxide (Sigma-Aldrich)).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

When needed, samples were thawed rapidly in a water bath (37 °C), then washed twice in R10 before downstream use.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Intestinal biopsy samples were collected at endoscopy from duodenum.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Biopsy samples for intestinal lymphocyte extraction were immediately placed in sterile R10 medium (as above) or MACS Tissue Storage Solution (Miltenyi Biotec) on ice for transportation, before cryopreservation in CryoStor Cs10 (STEMCELL Technologies).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

This approach preserves immune cell viability and surface marker expression.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

When required, samples were rapidly thawed in a 37 °C water bath and washed in 20 ml R10 before tissue dissociation.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

For immune cell isolation from duodenal biopsy samples for scRNA-seq (10x Genomics, dataset 1), biopsy samples were incubated in R10 medium with 1 mg ml Collagenase D (Roche) and 100 mg ml DNase (Thermo Fisher Scientific) for 1 h in a shaking incubator at 37 °C.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Biopsy samples were then dissociated by vigorous agitation using a GentleMACS Dissociator (Miltenyi Biotec), then strained through a 70-μm filter.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

The mononuclear cells were isolated on a discontinuous 70% and 35% Percoll gradient (GE Healthcare) by centrifugation at 700g for 20 min without brake.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Mononuclear cells were collected from the interface and washed in R10.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Cells were washed with R10 medium before antibody staining and downstream applications.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

The complete protocol with all steps is available at https://www.protocols.io/view/freezing-and-processing-intestinal-biopsies-for-th-dm6gp8745lzp/v1/ (Oxford HCA, 2019).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

The method for EC isolation from duodenal biopsy samples for scRNA-seq (10x Genomics, dataset 1) was adapted from ref. .

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Biopsy samples were washed in wash medium (HPGA, 1 mM EDTA, 1 mM dithiothreitol), then incubated in chelation medium (HPGA, 1 mM EDTA) at 37 °C for 40 min with agitation.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

The supernatant, which was removed and replaced every 10 min and contained epithelial crypts, was digested into a single-cell suspension by dissociation in a shaking incubator with TrypLE Express and DNase (50 µg ml) for 60 min at 37 °C.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

The epithelial single-cell suspension was washed with PBS and passed through a 30-µm filter.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Cell counts and viability were confirmed with a manual hemocytometer before further processing.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

The complete protocol with all steps is available at https://www.protocols.io/view/isolation-of-cells-from-the-epithelial-layer-of-fr-e6nvw9e6dgmk/v1/ (Oxford HCA, 2019).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

For immune cell isolation from duodenal biopsy samples for scRNA-seq and proteomics (BD Rhapsody, dataset 2), samples were thawed, then diluted with warm X-VIVO (Lonza) + 1% AB serum (Sigma-Aldrich).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Biopsy samples underwent enzymatic and mechanical digestion using 0.042 mg ml Liberase TL (Roche) and 1 mg ml DNAse I (Thermo Fisher).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Samples were placed horizontally in a shaking incubator for 20 min at 37 °C and homogenized using a gentleMACS Dissociator (Miltenyi Biotech).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Dissociated cells were passed through a 70-μm strainer.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Lymphocytes were enriched by Percoll density centrifugation as above.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Duodenal biopsy samples for flow cytometry were processed as for scRNA-seq of immune cells (see above).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Cells were washed with R10 medium before antibody staining and downstream applications.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Duodenal biopsy samples for fluorescence-activated cell sorting (FACS) of IELs for RNA-seq and TCR repertoire sequencing were placed in 10 ml HBSS with 1 mM EDTA and 1 mM dithiothreitol (both Sigma-Aldrich) and placed in a shaking incubator (200 rpm, 37 °C) for 15 min.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

IELs were strained through a 70-μm filter and washed two to three times with R10 before downstream applications.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

For spatial transcriptomics, single intestinal biopsy samples were embedded in OCT cryo-embedding matrix (Thermo Fisher Scientific) then frozen in isopentane (Sigma-Aldrich) suspended over liquid nitrogen or dry ice, and stored at −80 °C until use.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

For surface marker staining, cells were stained in 50 ml of FACS buffer (PBS + 1 mM EDTA + 0.05% BSA) for 30 min at 4 °C.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Surface antibodies and clones used are listed in Supplementary Table 3.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Antibodies were purchased from BioLegend, BD Biosciences, Miltenyi Biotec or Thermo Fisher Scientific.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

After staining, cells were stored at 4 °C protected from light until data acquisition.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Flow cytometry data were acquired on a BD LSR II flow cytometer (BD Biosciences).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

FACS samples were surface stained as above, with Sytox Green (Thermo Fisher Scientific) used as a viability dye.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

FACS was performed on an Aria III (BD Biosciences; 70-mm nozzle).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

For sorting by FACS for scRNA-seq of intestinal immune populations, cells were stained with EpCam-PE, CD27-BV421 and CD45-APC-Cy7.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Live CD45 or CD27 cells were sorted to include all mucosal immune cell populations, including long-lived CD27 plasma cells, which can downregulate surface CD45 expression.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

For sorting by FACS for scRNA-seq of intestinal epithelial populations, cells were stained with EpCAM-PE and CD45-AF700, with live EpCAM cells sorted.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

For sorting by FACS for bulk RNA-seq or TCR-seq of CD8 IEL populations, cells were stained with CD45-BV785, CD3-BV711, αβTCR-APC, γδTCR-PE, CD4-BV650 and CD8a-AF700, with live CD45CD3αβTCRCD8CD4 cells sorted.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Libraries were generated using 10x Genomics Chromium Single Cell V(D)J Reagents Kits (v1 Chemistry) per the manufacturer’s instructions.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Sorted cells suspended in PBS (plus 0.04% BSA) at a concentration of 1,000 cells per microliter were loaded into one lane of a Chromium controller.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Library quality and quantity were assessed using a TapeStation (Agilent) and Qubit Fluorometer (Thermo Fisher Scientific).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Libraries were sequenced on an Illumina HiSeq 4000 following the manufacturer’s instructions.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Library generation and sequencing were performed at the Sanger Institute, Cambridge.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Sorted CD45 cells were stained with a cocktail of 79 oligonucleotide-conjugated AbSeq antibodies (BD Biosciences, for 45 min at 4 °C.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Cells were then washed to remove residual unbound AbSeq antibodies and loaded onto three BD Rhapsody cartridges (BD Biosciences) for single-cell capture.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

AbSeq antibodies used in this study are listed in Supplementary Table 2.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Single-cell capture and cDNA library preparation were performed using the BD Rhapsody Express Single-Cell Analysis System (BD Biosciences), according to the manufacturer’s instructions.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Briefly, cDNA was amplified—ten cycles for resting cells and nine cycles for in vitro-stimulated cells—using the Human Immune Response Primer Panel (BD Biosciences; Supplementary Table 6), containing 399 primer pairs and a supplementary panel of 105 primer pairs (BD Biosciences; Supplementary Table 3).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

The resulting PCR1 products were purified using AMPure XP magnetic beads (Beckman Coulter), and the respective mRNA and AbSeq/Sample Tag products were separated based on size selection, using different bead ratios (0.7× and 1.2×, respectively).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

The purified mRNA and Sample Tag PCR1 products were further amplified (ten cycles), and the resulting PCR2 products purified by size selection (1× and 1.2× for the mRNA and Sample Tag libraries, respectively).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

The concentration, size and integrity of the resulting PCR products were assessed using both Qubit (High-Sensitivity dsDNA Kit; Thermo Fisher) and the Agilent 4200 TapeStation system (High Sensitivity D1000 ScreenTape; Agilent).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

The final products were normalized to 2.5 ng μl (mRNA), 0.5 ng μl (Sample Tag) and 0.275 ng μl (AbSeq) and underwent a final round of amplification (six cycles for mRNA and eight cycles for Sample Tag and AbSeq) using indexes for Illumina sequencing to prepare the final libraries.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Final libraries were quantified using Qubit and Agilent TapeStation and pooled (~60%/38%/2% mRNA/AbSeq/Sample Tag ratios, respectively) to achieve a final concentration of 5 nM. Final pooled libraries were spiked with 10% PhiX control DNA to increase sequence complexity and sequenced (75 base pairs (bp), paired-end) on a HiSeq 4000 sequencer (Illumina).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Cryopreserved, OCT-embedded duodenal biopsy samples were stored at −80 °C until use.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Before performing the full protocol, a tissue permeabilization optimization was performed (10x Genomics, Visium Spatial Tissue Optimization), which identified 11 min as the optimum permeabilization time.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Samples were processed for spatial transcriptomics per the manufacturer’s instructions (10x Genomics, Visium Spatial), with 2 × 10-μm sections cut on a pre-cooled cryostat for each sample onto two 6.5 × 6.5-mm capture areas, each with approximately 5,000 oligonucleotide-barcoded 55-μm-diameter spots.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Slides were fixed, H&E stained and imaged on a Leica DMI8 Widefield microscope at a magnification of ×40.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Tissue was permeabilized per instructions for 11 min, followed by reverse transcription and second-strand synthesis performed on the slide.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

cDNA quantification was performed using qPCR using KAPA SYBR FAST-qPCR kit (KAPA Biosystems) on a CFX96 Thermal Cycler instrument (Bio-Rad).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Following library construction per instructions, the spatial transcriptomics libraries were quantified and pooled at a concentration of 4 nM with a sample ratio corresponding to the approximate surface area of tissue coverage obtained from the H&E imaging.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Pooled libraries were sequenced on a NextSeq (Illumina) using a 150-bp paired-end dual-indexed setup (High output, v2.5, Illumina) loaded at a concentration of 1.8 pM, and sequenced to a manufacturer-recommended depth of a minimum of 50,000 reads per tissue-covered spot.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Cryopreserved, OCT-embedded duodenal biopsy samples were stored at −80 °C until use.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Spatial transcriptomics was performed using the Visium CytAssist (10x Genomics) workflow for fresh-frozen tissue according to the manufacturer’s instructions.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Ten-micron sections were cut on a cryostat pre-cooled to −20 °C and placed on 11 × 11-mm areas (four sections per area) on SuperFrost Plus slides (Thermo Fisher).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Sections were fixed, H&E stained and imaged on an Axioscan Z1 slide scanner (Zeiss) at a magnification of ×20.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Sections were de-stained and subjected to on-slide probe hybridization and ligation followed by probe transfer onto Visium CytAssist Spatial Gene Expression slides, each containing an 11 × 11-mm capture area covered by approximately 14,000 55-µm-diameter oligonucleotide-barcoded spots.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Probes were extended and cDNA quantified by qPCR using a KAPA SYBR FAST-qPCR kit (KAPA Biosystems) on a CFX96 Thermal Cycler instrument (Bio-Rad), followed by off-slide library construction per the instructions.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Libraries were quantified and pooled at a concentration of 2 nM with a sample ratio corresponding to the approximate surface area of tissue coverage obtained from the H&E imaging.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Pooled libraries were sequenced on a NextSeq 500 instrument (Illumina) using a 150-bp paired-end dual-indexed setup (High output, v2.5, Illumina) at a manufacturer-recommended depth of a minimum of 50,000 reads per tissue-covered spot.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

A TRIzol nucleic acid extraction method was used to extract RNA from low numbers of sorted lymphocytes, as previously described, except that phase-lock gel tubes were replaced with standard 1.5 ml microcentrifuge tubes.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Briefly, after sorting, cells were centrifuged (500g, 5 min), resuspended in 1 ml TRIzol, then frozen at −80 °C until RNA extraction.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

For RNA extraction, samples were thawed, mixed with 200 μl chloroform and centrifuged (14,000g, 5 min).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

A total of 500 μl of the aqueous phase was taken and RNA was extracted using the Agencourt RNAdvance Tissue Isolation kit.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

RNA concentration and purity were assessed on a 2100 Bioanalyzer instrument (Agilent).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Bulk RNA-seq was performed using the Smart-seq2 protocol at the Oxford Genomics Centre (University of Oxford).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Around 10 ng RNA was used as a template from each sample for library generation.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Barcoded samples were pooled, and External RNA Controls Consortium RNA (1:100,000 dilution) was added before 75-bp paired-end sequencing on an Illumina HiSeq 4000 instrument.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Bulk TCR repertoire sequencing was performed using the amplicon-rescued multiplex-PCR method (iRepertoire).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

This method performs an initial first-round RT–PCR with TCR V and C gene-specific primers for the relevant TCR chain, followed by further amplification steps with universal primers for the exponential phase of amplification.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

This method is designed to provide quantitative, deep sequencing of the TCR repertoire, with minimal bias.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Library generation was performed following the manufacturer’s instructions, except for using 96-well plates.

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

The quality, size distribution, concentration and presence of contaminating primer dimers of the final product was assessed using agarose gel electrophoresis, a spectral photometer (Nanodrop, Thermo Fisher Scientific), and the Bioanalyser DNA 1000 assay using a 2100 Bioanalyzer instrument (Agilent).

PMC12133578

Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.

Libraries were quantified using the KAPA Library Quantification Kit (Roche) on a CFX96 Thermal Cycler instrument (Bio-Rad) before equimolar pooling.