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| Status |
Public on Apr 30, 2023 |
| Title |
Primary cell population - EC |
| Sample type |
SRA |
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| Source name |
Lung
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| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague Dawley
cell type: Microvascular endothelial cells
age: 4-6 weeks
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| Growth protocol |
Fibroblasts (FB) were cultured 72 hrs on uncoated tissue-culture plastic in DMEM + 10% FBS + 1% Pen/Strep. Endothelial cells (ECs) were cultured to passage 5 on fibronectin-coated tissue-culture plastic in MCDB-131 Complete medium with serum (VEC Technologies)
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
AEC2: processed from fresh isolate. FB: trypsinized to single-cell suspension. EC: trypsinized to single-cell suspension. Native and engineered tissues: inflated with enzyme solution containing collagenase/dispase, elastase, and DNase and incubated for 20min, then sequentially filtered. Following digestion all steps were performed on ice. Total time from tissue inflation to library preparation was <2hrs.
Single-cell libraries were prepared using standard 10x Genomics protocols for v2 assay (for AEC2s and FBs), v3 assay (for native and engineered lung tissue), or v3.1 assay (for ECs)
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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 |
FASTQs were aligned, filtered, and counted using the count function in Cell Ranger (v3.0.2, 10x Genomics)
Subsequent data processing was done in R v3.6.1 using Seurat v3.1.1. Initial filtering accepted cells having 500-10,000 genes and 500-100,000 UMI, and percentage mitochondrial reads as follows: >1% and <7% for AEC2; <5% for FB; <9% for EC; <28% for P7; <25% for AEC2_FB_d7; and >6% and <28% for Tri-culture_d7
In Seurat, expression matrices were normalized using the NormalizeData function and scaled with ScaleData, with regression on percentage mitochondrial reads, number of UMI counts, and cell cycle scoring.
As an additional filtration step, following clustering, objects were subclustered by lineage (Col3a1+ mesenchyme, Epcam+ epithelium, Cdh5+ endothelium, and Ptprc+ immune), if present, to identify and remove suspected doublets and low-information cells. The filtered subsets were merged to create the objects for downstream processing.
Assembly: Ensembl Rnor6.0 release 95
Supplementary files format and content: matrix.mtx: sparse matrix file of raw counts, from Cell Ranger output
Supplementary files format and content: genes.tsv: row indices (gene names) of the raw count matrix, from Cell Ranger output
Supplementary files format and content: barcodes.tsv: column indices (cell barcodes) of the raw count matrix, from Cell Ranger output
Supplementary files format and content: data.txt: matrix of raw counts for every gene and cell in the final filtered object; rows are genenames, columns are cell barcodes
Supplementary files format and content: idents.tsv: cluster IDs for cells in the final filtered object; row names match cell barcodes (columns) in data.txt file
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| Submission date |
Feb 07, 2023 |
| Last update date |
May 02, 2023 |
| Contact name |
Katherine L Leiby |
| E-mail(s) |
katherine.leiby@yale.edu
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| Organization name |
Yale University
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| Department |
Biomedical Engineering
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| Lab |
Niklason Lab
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| Street address |
10 Amistad St. Rm 314
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| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06519 |
| Country |
USA |
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| Platform ID |
GPL25947 |
| Series (1) |
| GSE178405 |
Endothelial cells synergize with fibroblasts to form a niche for engineering alveolar epithelium |
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| Relations |
| SRA |
SRX19306106 |
| BioSample |
SAMN33246980 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7032062_EC.data.txt.gz |
15.6 Mb |
(ftp)(http) |
TXT |
| GSM7032062_EC.idents.tsv.gz |
20.2 Kb |
(ftp)(http) |
TSV |
| GSM7032062_EC_barcodes.tsv.gz |
19.7 Kb |
(ftp)(http) |
TSV |
| GSM7032062_EC_genes.tsv.gz |
201.2 Kb |
(ftp)(http) |
TSV |
| GSM7032062_EC_matrix.mtx.gz |
63.0 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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