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| Status |
Public on Mar 21, 2023 |
| Title |
Spatial CLAD sample 2 |
| Sample type |
SRA |
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| Source name |
Lung
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Lung
cell line: Primary lung tissue
condition: CLAD
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| Extracted molecule |
total RNA |
| Extraction protocol |
A 2cm x 2cm segment of lung tissue was isolated at least 1cm from the pleura and minced using a razor blade. Tissue was digested at 37C using Miltenyi Multi Tissue Dissociation Kit 1 (Miltenyi Biotech, 130-110-201) for 30 minutes. Mechanical digestion was performed three times using Miltenyi gentleMACS dissociator (Miltenyi, 130-093-235). The cell suspension was spun down at 300g for 10 minutes, and cells were treated with RBC lysis buffer (Sigma, R7757). The cell suspension was filtered using a 70μm filter, spun down at 300g for 10 minutes, and resuspended in PBS with 0.04% BSA. For spatial transcriptomics, 10µm sections were cut from frozen OCT blocks and mounted onto Visium spatial gene expression slides and processed according to the manufacturer’s protocol (10x Genomics). Tissue sections were permeabilized immediately to capture RNA molecules onto barcoded spots on the slide.
Single cells were prepared for cDNA amplification and library preparation using Single Cell 3’ Reagent Kits v2 (10x Genomics) following the manufacturer's protocol
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10x Genomics
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| Data processing |
Cell Ranger v5.0.1 was used to demultiplex raw FASTQ files, align the reads to the 10x GRCh38 reference transcriptome, and generate gene expression matrices for all single cells in each sample. Further downstream analysis was performed using the R package, Seurat v4.1.0.
The top 3,000 genes were selected based on variance using the Seurat implementation FindVariableFeatures. These genes were then centered and scaled using Seurat’s implementation ScaleData and percent mitochondrial genes as a variable to regress out (percent_mt = 0.25). Scaled data was subsequently used for principal components analysis (npcs = 42), and cell cluster analysis was performed using Seurat’s FindNeighbors (dims = 1:42) and FindClusters (resolution = 1.2) functions. Visualization was performed using uniform manifold approximation and projection.
Fiducial 50µm spots on the H&E image were manually aligned using Loupe browser v5.0.1 (10x Genomics). Space Ranger software (10x Genomics) was used to process raw FASTQ files, align the reads to the 10x GRCh38 reference transcriptome, and generate gene expression matrices for all single cells in each sample. Further downstream analysis of the spot UMI table and reference H&E section was performed in R using Seurat v4.1.0.
Assembly: GRCh38
Supplementary files format and content: Tab-delimited files of single cell dataset include (1) gene expression matrix following sctransform based normalization and (2) cell identities including barcodes, CLAD vs control disease annotation, sample ID, and cell type annotations.
Supplementary files format and content: Loupe files for spatial dataset
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| Submission date |
Feb 01, 2023 |
| Last update date |
Mar 22, 2023 |
| Contact name |
Scott Palmer |
| E-mail(s) |
scott.palmer@duke.edu
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| Organization name |
Duke University
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| Street address |
2100 Erwin Road
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| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27705 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE224210 |
Single cell and spatial transcriptomic profile of whole CLAD and donor control lung tissue |
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| Relations |
| BioSample |
SAMN33005012 |
| SRA |
SRX19243997 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7017458_ST_CLAD_2.cloupe.gz |
169.5 Mb |
(ftp)(http) |
CLOUPE |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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