Mardiyyah/TAPT_data_V2_split · Datasets at Hugging Face

PMCID stringclasses 24 values	Title stringclasses 24 values	Sentences stringlengths 2 40.7k
PMC10287567	An integrated cell atlas of the lung in health and disease.	The data were derived from the clinical trial registered at ClinicalTrials.gov under reference NCT04529993.
PMC10287567	An integrated cell atlas of the lung in health and disease.	This study was described as an interventional study instead of an observational study because the participants were volunteers and all assigned to a specific bronchoscopy not related to routine medical care.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Participants were prospectively assigned to a procedure (bronchoscopy) according to a specific protocol to assess our ability to sample the airway.
PMC10287567	An integrated cell atlas of the lung in health and disease.	No other procedures were included in this study.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Metadata of the donors’ sex was based on self-report.
PMC10287567	An integrated cell atlas of the lung in health and disease.	The libraries were prepared as described in Deprez et al. and yielded an average of 61,000 ± 11,000 cells per sample, with a viability above 95%.
PMC10287567	An integrated cell atlas of the lung in health and disease.	The single-cell suspension was used to generate single-cell libraries following the v3.1 protocol for 3′ chemistry from 10x Genomics (CG000204).
PMC10287567	An integrated cell atlas of the lung in health and disease.	Sequencing was performed on a NextSeq 500/550 sequencer (Illumina).
PMC10287567	An integrated cell atlas of the lung in health and disease.	Raw sequencing data were processed using the Cell Ranger 6.0.0 pipeline, with the reference genome GRCh38 and annotation using Ensembl98.
PMC10287567	An integrated cell atlas of the lung in health and disease.	For each sample, cells with fewer than 200 transcripts or more than 40,000 transcripts were filtered out, as well as genes expressed in fewer than three cells.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Normalization and log transformation were done using the standard Scanpy pipeline.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Principal component analysis (PCA) was performed on 1,000 highly variable genes (HVGs) to compute 50 principal components, and the Louvain algorithm was used for clustering.
PMC10287567	An integrated cell atlas of the lung in health and disease.	These clusters were then annotated by hand for each sample.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Raw counts and the thus obtained cell annotations were used as input for the HLCA.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Tumor-free, uninvolved lung samples (peritumor tissues) were obtained during tumor resections at the lung specialist clinic Asklepios Fachkliniken München-Gauting and accessed through the bioArchive of the Comprehensive Pneumology Center in Munich.
PMC10287567	An integrated cell atlas of the lung in health and disease.	The study was approved by the local ethics committee of the Ludwig Maximilian University of Munich (EK 333-10 and 382-10), and written informed consent was obtained from all patients.
PMC10287567	An integrated cell atlas of the lung in health and disease.	All fresh tissues from patients in a given time frame without any specific selection criteria were included, and only patients with obvious chronic lung disease as comorbidity based on their lung function parameters before tumor resection were excluded.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Metadata of the donors’ sex were based on self-report.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Single-cell suspensions for scRNA-seq were generated as previously described.
PMC10287567	An integrated cell atlas of the lung in health and disease.	In brief, lung tissue samples were cut into smaller pieces, washed with phosphate-buffered saline (PBS) and enzymatically digested using an enzyme mix composed of dispase, collagenase, elastase and DNAse for 45 min at 37 °C while shaking.
PMC10287567	An integrated cell atlas of the lung in health and disease.	After inactivating the enzymatic activity with 10% fetal calf serum (FCS)/PBS, dissociated cells were passed through a 70 µm cell strainer, pelleted by centrifugation (300g; 5 min) and subjected to red blood cell lysis.
PMC10287567	An integrated cell atlas of the lung in health and disease.	After stopping the lysis with 10% FCS/PBS, the cell suspension was passed through a 30 µm strainer and pelleted.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Cells were resuspended in 10% FCS/PBS, assessed for viability and counted using a Neubauer hematocytometer.
PMC10287567	An integrated cell atlas of the lung in health and disease.	The cell concentration was adjusted to 1,000 cells per µl and ~16,000 cells were loaded on a 10x Genomics Chip G with Chromium Single Cell 3′ v3.1 gel beads and reagents (3′ GEX v3.1; 10x Genomics).
PMC10287567	An integrated cell atlas of the lung in health and disease.	Libraries were prepared according to the manufacturer’s protocol (CG000204_RevD; 10× Genomics).
PMC10287567	An integrated cell atlas of the lung in health and disease.	After a quality check, scRNA-seq libraries were pooled and sequenced on a NovaSeq 6000 instrument.
PMC10287567	An integrated cell atlas of the lung in health and disease.	The generation of count matrices was performed using the Cell Ranger computational pipeline (v3.1.0; STAR v2.5.3a).
PMC10287567	An integrated cell atlas of the lung in health and disease.	The reads were aligned to the GRCh38 human reference genome (GRCh38; Ensembl99).
PMC10287567	An integrated cell atlas of the lung in health and disease.	Downstream analysis was performed using the Scanpy package (version 1.8.0).
PMC10287567	An integrated cell atlas of the lung in health and disease.	We assessed the quality of our libraries and excluded barcodes with fewer than 300 genes detected, while retaining those with a number of transcripts between 500 and 30,000.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Furthermore, cells with a high proportion (>15%) of transcript counts derived from mitochondrial-encoded genes were removed.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Genes were considered if they were expressed in at least five cells.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Raw counts of cells that passed filtering were used as input for the HLCA.
PMC10287567	An integrated cell atlas of the lung in health and disease.	All postmortem human donor lung samples were obtained from BRINDL, supported by the NHLBI LungMAP Human Tissue Core housed at the University of Rochester.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Consent, tissue acquisition and storage protocols can be found on the repository’s website (brindl.urmc.rochester.edu/).
PMC10287567	An integrated cell atlas of the lung in health and disease.	Data were collected as part of the Human Biomolecular Atlas Program (HuBMAP).
PMC10287567	An integrated cell atlas of the lung in health and disease.	Metadata of the donor’s sex were based on self-report.
PMC10287567	An integrated cell atlas of the lung in health and disease.	For isolation of single nuclei, ten cryosections (40 µm thickness) from O.C.T.-embedded tissue blocks stored at −80 °C were shipped on dry ice and processed according to a published protocol.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Single-nucleus RNA-seq was completed using 10x Chromium Single Cell 3’ Reagent Kits v3, according to a published protocol.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Raw sequencing data were processed using the 10x Cell Ranger v3 pipeline and the GRCh38 reference genome.
PMC10287567	An integrated cell atlas of the lung in health and disease.	For downstream analysis, mitochondrial transcripts and doublets identified by DoubletDetection version 2.4.0 were removed.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Samples were then combined and cell barcodes were filtered based on the genes detected (>200 and <7,500) and the gene unique molecular identifier (UMI) ratio (gene.vs.molecule.cell.filter function) using Pagoda2 (github.com/hms-dbmi/pagoda2).
PMC10287567	An integrated cell atlas of the lung in health and disease.	Also using Pagoda2 for clustering, counts were normalized to total counts per nucleus.
PMC10287567	An integrated cell atlas of the lung in health and disease.	For batch correction, gene expression was scaled to dataset average expression.
PMC10287567	An integrated cell atlas of the lung in health and disease.	After variance normalization, all significantly variant genes (n = 4,519) were used for PCA.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Clustering was done at different k values (50, 100 or 200) using the top 50 principal components and the infomap community detection algorithm.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Then, principal component and cluster annotations were imported into Seurat version 4.0.0.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Differentially expressed genes for all clusters were generated for each k resolution using Seurat FindAllMarkers (only.pos = TRUE, max.cells.per.ident = 1000, logfc.threshold = 0.25, min.pct = 0.25).
PMC10287567	An integrated cell atlas of the lung in health and disease.	Clusters were manually annotated based on distinct differentially expressed marker genes.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Raw counts and the thus obtained cell annotations were used as input for the HLCA.
PMC10287567	An integrated cell atlas of the lung in health and disease.	These data were a combination of published and unpublished data.
PMC10287567	An integrated cell atlas of the lung in health and disease.	In both cases, healthy volunteers were recruited for bronchoscopy at the University Medical Center in Groningen after giving informed consent and according to the protocol approved by the IRB (ABR number NL69765.042.19).
PMC10287567	An integrated cell atlas of the lung in health and disease.	Inclusion criteria and tissue processing were performed as previously described.
PMC10287567	An integrated cell atlas of the lung in health and disease.	In short, all donors were 20–65 years old and had a history of smoking <10 pack-years.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Metadata of the donors’ sex were based on self-report.
PMC10287567	An integrated cell atlas of the lung in health and disease.	To exclude respiratory disease, the following criteria were used: absent history of asthma or COPD; no use of asthma- or COPD-related medication; a negative provocation test (concentration of methacholine that provokes a 20% decrease in the forced expiratory volume in 1 s (FEV1) > 8 mg ml); no airflow obstruction (FEV1/forced vital capacity ≥ 70%); and an absence of lung function impairment (that is, FEV1 ≥ 80% predicted).
PMC10287567	An integrated cell atlas of the lung in health and disease.	All donors underwent a bronchoscopy under sedation using a standardized protocol.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Nasal brushes were obtained from the lateral inferior turbinate in a subset of the volunteers immediately before bronchoscopy using a Cyto-Pak CytoSoft nasal brush (Medical Packaging Corporation).
PMC10287567	An integrated cell atlas of the lung in health and disease.	Six macroscopically adequate endobronchial biopsies were collected for this study, located between the third and sixth generation of the right lower and middle lobe.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Bronchial brushes were obtained from a different airway at similar anatomical locations using a Cellebrity bronchial brush (Boston Scientific).
PMC10287567	An integrated cell atlas of the lung in health and disease.	Extracted biopsies and bronchial and nasal brushes were processed directly, with a maximum of 1 h delay.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Bronchial biopsies were chopped biopsies using a single-edge razor blade.
PMC10287567	An integrated cell atlas of the lung in health and disease.	A single-cell solution was obtained by tissue digestion using 1 mg ml collagenase D and 0.1 mg ml DNase I (Roche) in Hanks’ Balanced Salt Solution (Lonza) at 37 °C for 1 h with gentle agitation for both nasal brushes and bronchial biopsies.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Single-cell suspensions were filtered and forced using a 70 µm nylon cell strainer (Falcon), followed by centrifugation at 550g and 4 °C for 5 min and one wash with PBS containing 1% bovine serum albumin (BSA; Sigma–Aldrich).
PMC10287567	An integrated cell atlas of the lung in health and disease.	The single-cell suspensions used for 10x Genomics scRNA-seq analysis were cleared of red blood cells using a red blood cell lysis buffer (eBioscience) followed by live cell counting and loading of 10,000 cells per lane.
PMC10287567	An integrated cell atlas of the lung in health and disease.	We used 10x Genomics Chromium Single Cell 3′ Reagent Kits v2 and v3 according to the manufacturers’ instructions.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Raw sequencing data were processed using the Cell Ranger 3.1.0-based HLCA pipeline, with the reference genome GRCh38 and annotation using Ensembl98.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Ambient RNA correction was performed with FastCAR, using an empty library cutoff of 100 UMI and a maximum allowed contamination chance of 0.05, ignoring the mitochondrial RNA.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Data were merged and processed using Seurat, filtering to libraries with >500 UMIs and >200 genes and to the libraries containing the lowest 95% of mitochondrial RNA per sample and <25% mitochondrial RNA, normalized using sctransform while regressing out variation correlating with the percentage of mitochondrial RNA per cell.
PMC10287567	An integrated cell atlas of the lung in health and disease.	In general, 15 principal components were used for the clustering, at a resolution of 0.5 to facilitate manual annotation of the dataset.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Clusters in the final object that were driven by single donors were removed.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Raw counts and cell annotations were used as input for the HLCA.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Nasal epithelial samples were collected from healthy volunteers who provided informed consent at Northwestern Medicine in Chicago.
PMC10287567	An integrated cell atlas of the lung in health and disease.	The protocol was approved by the Northwestern University IRB (STU00214826).
PMC10287567	An integrated cell atlas of the lung in health and disease.	Healthy volunteers were recruited to match a cohort of patients with cystic fibrosis for the ongoing study at Northwestern University (with M.J. as the principal investigator).
PMC10287567	An integrated cell atlas of the lung in health and disease.	In both studies, A.V.M. did not influence participant recruitment and did not introduce biases in sample selection.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Metadata of the donors’ sex were based on self-report.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Briefly, donors were seated and asked to extend their neck.
PMC10287567	An integrated cell atlas of the lung in health and disease.	A nasal curette (Rhino-Pro; VWR) was inserted into either nare and gently slid in the direction of posterior to anterior ~1 cm along the lateral inferior turbinate.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Five curettes were obtained per participant.
PMC10287567	An integrated cell atlas of the lung in health and disease.	The curette tip was then cut and placed in 2 ml hypothermosol and stored at 4 C until processing.
PMC10287567	An integrated cell atlas of the lung in health and disease.	A single-cell suspension was generated using the cold-active dispase protocol reported by Deprez et al. and Zaragosi and Barbry with slight modification.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Specifically, ethylenediaminetetraacetic acid (EDTA) was omitted and cells were dispersed by pipetting 20 times every 5 min using a 1 ml tip instead of tritration using a 21/23 G needle.
PMC10287567	An integrated cell atlas of the lung in health and disease.	The final concentration of protease from Bacillus licheniformis was 10 mg ml.
PMC10287567	An integrated cell atlas of the lung in health and disease.	The total digestion time was 30 min.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Following the wash in 4 ml 0.5% BSA in PBS and centrifugation at 400g for 10 min, cells were resuspended in 0.5% BSA in PBS and counted using a Nexcelom K2 Cellometer with acridine orange/propidium iodide reagent.
PMC10287567	An integrated cell atlas of the lung in health and disease.	This protocol typically yields ~300–500,000 cells with a viability of >95%.
PMC10287567	An integrated cell atlas of the lung in health and disease.	The resulting single-cell suspension was then used to generate single-cell libraries following the protocol for 5′ V1 (CG000086 Rev M; 10x Genomics) or V2 chemistry (CG000331 Rev A; 10x Genomics).
PMC10287567	An integrated cell atlas of the lung in health and disease.	Excess cells from two of the samples were pooled together to generate one additional single-cell library.
PMC10287567	An integrated cell atlas of the lung in health and disease.	After a quality check, the libraries were pooled and sequenced on a NovaSeq 6000 instrument.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Raw sequencing data were processed using the Cell Ranger 3.1.0 pipeline, with the reference genome GRCh38 and annotation using Ensembl98.
PMC10287567	An integrated cell atlas of the lung in health and disease.	To assign sample information to cells in the single-cell library prepared from two samples, we ran souporcell version 2.0 for that library and two libraries that were prepared from these samples separately.
PMC10287567	An integrated cell atlas of the lung in health and disease.	We used common genetic variants prepared by the souporcell authors to separate cells into two groups by genotype for each library, and Pearson correlation between the identified genotypes across libraries to establish correspondence between genotype and sample.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Cell annotations were assigned to cell clusters based on expert interpretation of marker genes for each cluster.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Cell clusters were derived with the Seurat version 3.2 workflow in which samples were normalized with sctransform, 3,000 HVGs were selected and integrated and clusters were derived from 30 principal components using the Louvain algorithm with default parameters.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Clusters with a low number of UMIs and high expression of ribosomal or mitochondrial genes were excluded as low quality.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Raw counts and the thus obtained cell annotations were used as input for the HLCA.
PMC10287567	An integrated cell atlas of the lung in health and disease.	Human lung tissue wabus available for research purposes following ethical approval from Hannover Medical School (Nr.
PMC10287567	An integrated cell atlas of the lung in health and disease.	7414, 2017).
PMC10287567	An integrated cell atlas of the lung in health and disease.	All patients in this study provided written informed consent for sample collection and data analyses.

PMC10287567

An integrated cell atlas of the lung in health and disease.

The data were derived from the clinical trial registered at ClinicalTrials.gov under reference NCT04529993.

PMC10287567

An integrated cell atlas of the lung in health and disease.

This study was described as an interventional study instead of an observational study because the participants were volunteers and all assigned to a specific bronchoscopy not related to routine medical care.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Participants were prospectively assigned to a procedure (bronchoscopy) according to a specific protocol to assess our ability to sample the airway.

PMC10287567

An integrated cell atlas of the lung in health and disease.

No other procedures were included in this study.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Metadata of the donors’ sex was based on self-report.

PMC10287567

An integrated cell atlas of the lung in health and disease.

The libraries were prepared as described in Deprez et al. and yielded an average of 61,000 ± 11,000 cells per sample, with a viability above 95%.

PMC10287567

An integrated cell atlas of the lung in health and disease.

The single-cell suspension was used to generate single-cell libraries following the v3.1 protocol for 3′ chemistry from 10x Genomics (CG000204).

PMC10287567

An integrated cell atlas of the lung in health and disease.

Sequencing was performed on a NextSeq 500/550 sequencer (Illumina).

PMC10287567

An integrated cell atlas of the lung in health and disease.

Raw sequencing data were processed using the Cell Ranger 6.0.0 pipeline, with the reference genome GRCh38 and annotation using Ensembl98.

PMC10287567

An integrated cell atlas of the lung in health and disease.

For each sample, cells with fewer than 200 transcripts or more than 40,000 transcripts were filtered out, as well as genes expressed in fewer than three cells.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Normalization and log transformation were done using the standard Scanpy pipeline.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Principal component analysis (PCA) was performed on 1,000 highly variable genes (HVGs) to compute 50 principal components, and the Louvain algorithm was used for clustering.

PMC10287567

An integrated cell atlas of the lung in health and disease.

These clusters were then annotated by hand for each sample.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Raw counts and the thus obtained cell annotations were used as input for the HLCA.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Tumor-free, uninvolved lung samples (peritumor tissues) were obtained during tumor resections at the lung specialist clinic Asklepios Fachkliniken München-Gauting and accessed through the bioArchive of the Comprehensive Pneumology Center in Munich.

PMC10287567

An integrated cell atlas of the lung in health and disease.

The study was approved by the local ethics committee of the Ludwig Maximilian University of Munich (EK 333-10 and 382-10), and written informed consent was obtained from all patients.

PMC10287567

An integrated cell atlas of the lung in health and disease.

All fresh tissues from patients in a given time frame without any specific selection criteria were included, and only patients with obvious chronic lung disease as comorbidity based on their lung function parameters before tumor resection were excluded.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Metadata of the donors’ sex were based on self-report.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Single-cell suspensions for scRNA-seq were generated as previously described.

PMC10287567

An integrated cell atlas of the lung in health and disease.

In brief, lung tissue samples were cut into smaller pieces, washed with phosphate-buffered saline (PBS) and enzymatically digested using an enzyme mix composed of dispase, collagenase, elastase and DNAse for 45 min at 37 °C while shaking.

PMC10287567

An integrated cell atlas of the lung in health and disease.

After inactivating the enzymatic activity with 10% fetal calf serum (FCS)/PBS, dissociated cells were passed through a 70 µm cell strainer, pelleted by centrifugation (300g; 5 min) and subjected to red blood cell lysis.

PMC10287567

An integrated cell atlas of the lung in health and disease.

After stopping the lysis with 10% FCS/PBS, the cell suspension was passed through a 30 µm strainer and pelleted.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Cells were resuspended in 10% FCS/PBS, assessed for viability and counted using a Neubauer hematocytometer.

PMC10287567

An integrated cell atlas of the lung in health and disease.

The cell concentration was adjusted to 1,000 cells per µl and ~16,000 cells were loaded on a 10x Genomics Chip G with Chromium Single Cell 3′ v3.1 gel beads and reagents (3′ GEX v3.1; 10x Genomics).

PMC10287567

An integrated cell atlas of the lung in health and disease.

Libraries were prepared according to the manufacturer’s protocol (CG000204_RevD; 10× Genomics).

PMC10287567

An integrated cell atlas of the lung in health and disease.

After a quality check, scRNA-seq libraries were pooled and sequenced on a NovaSeq 6000 instrument.

PMC10287567

An integrated cell atlas of the lung in health and disease.

The generation of count matrices was performed using the Cell Ranger computational pipeline (v3.1.0; STAR v2.5.3a).

PMC10287567

An integrated cell atlas of the lung in health and disease.

The reads were aligned to the GRCh38 human reference genome (GRCh38; Ensembl99).

PMC10287567

An integrated cell atlas of the lung in health and disease.

Downstream analysis was performed using the Scanpy package (version 1.8.0).

PMC10287567

An integrated cell atlas of the lung in health and disease.

We assessed the quality of our libraries and excluded barcodes with fewer than 300 genes detected, while retaining those with a number of transcripts between 500 and 30,000.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Furthermore, cells with a high proportion (>15%) of transcript counts derived from mitochondrial-encoded genes were removed.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Genes were considered if they were expressed in at least five cells.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Raw counts of cells that passed filtering were used as input for the HLCA.

PMC10287567

An integrated cell atlas of the lung in health and disease.

All postmortem human donor lung samples were obtained from BRINDL, supported by the NHLBI LungMAP Human Tissue Core housed at the University of Rochester.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Consent, tissue acquisition and storage protocols can be found on the repository’s website (brindl.urmc.rochester.edu/).

PMC10287567

An integrated cell atlas of the lung in health and disease.

Data were collected as part of the Human Biomolecular Atlas Program (HuBMAP).

PMC10287567

An integrated cell atlas of the lung in health and disease.

Metadata of the donor’s sex were based on self-report.

PMC10287567

An integrated cell atlas of the lung in health and disease.

For isolation of single nuclei, ten cryosections (40 µm thickness) from O.C.T.-embedded tissue blocks stored at −80 °C were shipped on dry ice and processed according to a published protocol.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Single-nucleus RNA-seq was completed using 10x Chromium Single Cell 3’ Reagent Kits v3, according to a published protocol.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Raw sequencing data were processed using the 10x Cell Ranger v3 pipeline and the GRCh38 reference genome.

PMC10287567

An integrated cell atlas of the lung in health and disease.

For downstream analysis, mitochondrial transcripts and doublets identified by DoubletDetection version 2.4.0 were removed.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Samples were then combined and cell barcodes were filtered based on the genes detected (>200 and <7,500) and the gene unique molecular identifier (UMI) ratio (gene.vs.molecule.cell.filter function) using Pagoda2 (github.com/hms-dbmi/pagoda2).

PMC10287567

An integrated cell atlas of the lung in health and disease.

Also using Pagoda2 for clustering, counts were normalized to total counts per nucleus.

PMC10287567

An integrated cell atlas of the lung in health and disease.

For batch correction, gene expression was scaled to dataset average expression.

PMC10287567

An integrated cell atlas of the lung in health and disease.

After variance normalization, all significantly variant genes (n = 4,519) were used for PCA.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Clustering was done at different k values (50, 100 or 200) using the top 50 principal components and the infomap community detection algorithm.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Then, principal component and cluster annotations were imported into Seurat version 4.0.0.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Differentially expressed genes for all clusters were generated for each k resolution using Seurat FindAllMarkers (only.pos = TRUE, max.cells.per.ident = 1000, logfc.threshold = 0.25, min.pct = 0.25).

PMC10287567

An integrated cell atlas of the lung in health and disease.

Clusters were manually annotated based on distinct differentially expressed marker genes.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Raw counts and the thus obtained cell annotations were used as input for the HLCA.

PMC10287567

An integrated cell atlas of the lung in health and disease.

These data were a combination of published and unpublished data.

PMC10287567

An integrated cell atlas of the lung in health and disease.

In both cases, healthy volunteers were recruited for bronchoscopy at the University Medical Center in Groningen after giving informed consent and according to the protocol approved by the IRB (ABR number NL69765.042.19).

PMC10287567

An integrated cell atlas of the lung in health and disease.

Inclusion criteria and tissue processing were performed as previously described.

PMC10287567

An integrated cell atlas of the lung in health and disease.

In short, all donors were 20–65 years old and had a history of smoking <10 pack-years.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Metadata of the donors’ sex were based on self-report.

PMC10287567

An integrated cell atlas of the lung in health and disease.

To exclude respiratory disease, the following criteria were used: absent history of asthma or COPD; no use of asthma- or COPD-related medication; a negative provocation test (concentration of methacholine that provokes a 20% decrease in the forced expiratory volume in 1 s (FEV1) > 8 mg ml); no airflow obstruction (FEV1/forced vital capacity ≥ 70%); and an absence of lung function impairment (that is, FEV1 ≥ 80% predicted).

PMC10287567

An integrated cell atlas of the lung in health and disease.

All donors underwent a bronchoscopy under sedation using a standardized protocol.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Nasal brushes were obtained from the lateral inferior turbinate in a subset of the volunteers immediately before bronchoscopy using a Cyto-Pak CytoSoft nasal brush (Medical Packaging Corporation).

PMC10287567

An integrated cell atlas of the lung in health and disease.

Six macroscopically adequate endobronchial biopsies were collected for this study, located between the third and sixth generation of the right lower and middle lobe.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Bronchial brushes were obtained from a different airway at similar anatomical locations using a Cellebrity bronchial brush (Boston Scientific).

PMC10287567

An integrated cell atlas of the lung in health and disease.

Extracted biopsies and bronchial and nasal brushes were processed directly, with a maximum of 1 h delay.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Bronchial biopsies were chopped biopsies using a single-edge razor blade.

PMC10287567

An integrated cell atlas of the lung in health and disease.

A single-cell solution was obtained by tissue digestion using 1 mg ml collagenase D and 0.1 mg ml DNase I (Roche) in Hanks’ Balanced Salt Solution (Lonza) at 37 °C for 1 h with gentle agitation for both nasal brushes and bronchial biopsies.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Single-cell suspensions were filtered and forced using a 70 µm nylon cell strainer (Falcon), followed by centrifugation at 550g and 4 °C for 5 min and one wash with PBS containing 1% bovine serum albumin (BSA; Sigma–Aldrich).

PMC10287567

An integrated cell atlas of the lung in health and disease.

The single-cell suspensions used for 10x Genomics scRNA-seq analysis were cleared of red blood cells using a red blood cell lysis buffer (eBioscience) followed by live cell counting and loading of 10,000 cells per lane.

PMC10287567

An integrated cell atlas of the lung in health and disease.

We used 10x Genomics Chromium Single Cell 3′ Reagent Kits v2 and v3 according to the manufacturers’ instructions.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Raw sequencing data were processed using the Cell Ranger 3.1.0-based HLCA pipeline, with the reference genome GRCh38 and annotation using Ensembl98.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Ambient RNA correction was performed with FastCAR, using an empty library cutoff of 100 UMI and a maximum allowed contamination chance of 0.05, ignoring the mitochondrial RNA.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Data were merged and processed using Seurat, filtering to libraries with >500 UMIs and >200 genes and to the libraries containing the lowest 95% of mitochondrial RNA per sample and <25% mitochondrial RNA, normalized using sctransform while regressing out variation correlating with the percentage of mitochondrial RNA per cell.

PMC10287567

An integrated cell atlas of the lung in health and disease.

In general, 15 principal components were used for the clustering, at a resolution of 0.5 to facilitate manual annotation of the dataset.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Clusters in the final object that were driven by single donors were removed.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Raw counts and cell annotations were used as input for the HLCA.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Nasal epithelial samples were collected from healthy volunteers who provided informed consent at Northwestern Medicine in Chicago.

PMC10287567

An integrated cell atlas of the lung in health and disease.

The protocol was approved by the Northwestern University IRB (STU00214826).

PMC10287567

An integrated cell atlas of the lung in health and disease.

Healthy volunteers were recruited to match a cohort of patients with cystic fibrosis for the ongoing study at Northwestern University (with M.J. as the principal investigator).

PMC10287567

An integrated cell atlas of the lung in health and disease.

In both studies, A.V.M. did not influence participant recruitment and did not introduce biases in sample selection.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Metadata of the donors’ sex were based on self-report.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Briefly, donors were seated and asked to extend their neck.

PMC10287567

An integrated cell atlas of the lung in health and disease.

A nasal curette (Rhino-Pro; VWR) was inserted into either nare and gently slid in the direction of posterior to anterior ~1 cm along the lateral inferior turbinate.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Five curettes were obtained per participant.

PMC10287567

An integrated cell atlas of the lung in health and disease.

The curette tip was then cut and placed in 2 ml hypothermosol and stored at 4 C until processing.

PMC10287567

An integrated cell atlas of the lung in health and disease.

A single-cell suspension was generated using the cold-active dispase protocol reported by Deprez et al. and Zaragosi and Barbry with slight modification.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Specifically, ethylenediaminetetraacetic acid (EDTA) was omitted and cells were dispersed by pipetting 20 times every 5 min using a 1 ml tip instead of tritration using a 21/23 G needle.

PMC10287567

An integrated cell atlas of the lung in health and disease.

The final concentration of protease from Bacillus licheniformis was 10 mg ml.

PMC10287567

An integrated cell atlas of the lung in health and disease.

The total digestion time was 30 min.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Following the wash in 4 ml 0.5% BSA in PBS and centrifugation at 400g for 10 min, cells were resuspended in 0.5% BSA in PBS and counted using a Nexcelom K2 Cellometer with acridine orange/propidium iodide reagent.

PMC10287567

An integrated cell atlas of the lung in health and disease.

This protocol typically yields ~300–500,000 cells with a viability of >95%.

PMC10287567

An integrated cell atlas of the lung in health and disease.

The resulting single-cell suspension was then used to generate single-cell libraries following the protocol for 5′ V1 (CG000086 Rev M; 10x Genomics) or V2 chemistry (CG000331 Rev A; 10x Genomics).

PMC10287567

An integrated cell atlas of the lung in health and disease.

Excess cells from two of the samples were pooled together to generate one additional single-cell library.

PMC10287567

An integrated cell atlas of the lung in health and disease.

After a quality check, the libraries were pooled and sequenced on a NovaSeq 6000 instrument.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Raw sequencing data were processed using the Cell Ranger 3.1.0 pipeline, with the reference genome GRCh38 and annotation using Ensembl98.

PMC10287567

An integrated cell atlas of the lung in health and disease.

To assign sample information to cells in the single-cell library prepared from two samples, we ran souporcell version 2.0 for that library and two libraries that were prepared from these samples separately.

PMC10287567

An integrated cell atlas of the lung in health and disease.

We used common genetic variants prepared by the souporcell authors to separate cells into two groups by genotype for each library, and Pearson correlation between the identified genotypes across libraries to establish correspondence between genotype and sample.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Cell annotations were assigned to cell clusters based on expert interpretation of marker genes for each cluster.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Cell clusters were derived with the Seurat version 3.2 workflow in which samples were normalized with sctransform, 3,000 HVGs were selected and integrated and clusters were derived from 30 principal components using the Louvain algorithm with default parameters.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Clusters with a low number of UMIs and high expression of ribosomal or mitochondrial genes were excluded as low quality.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Raw counts and the thus obtained cell annotations were used as input for the HLCA.

PMC10287567

An integrated cell atlas of the lung in health and disease.

Human lung tissue wabus available for research purposes following ethical approval from Hannover Medical School (Nr.

PMC10287567

An integrated cell atlas of the lung in health and disease.

7414, 2017).

PMC10287567

An integrated cell atlas of the lung in health and disease.

All patients in this study provided written informed consent for sample collection and data analyses.