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| Status |
Public on Mar 08, 2021 |
| Title |
Control E16.5 scRNAseq |
| Sample type |
SRA |
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| Source name |
Lung epithelial, immune, endothelial, and mesenchymal cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: epithelial, immune, endothelial, and mesenchymal cells
developmental stage: Embryonic day 16.5
genotype: control
treatment: none
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| Extracted molecule |
total RNA |
| Extraction protocol |
Perfused lungs were collected from anesthetized mice as described above. Previously published protocols for cell dissociation and sorting cells were used with minor modifications11. Extra-pulmonary airways and connective tissues were removed from lungs, which were then minced with forceps and digested in Liebovitz media (Gibco, 21083-027) for scRNA-seq, ATAC-seq, or scATAC-seq with 2 mg/mL collagenase type I (Worthington, CLS-1, LS004197), 0.5 mg/mL DNase I (Worthington, D, LS002007), and 2 mg/mL elastase (Worthingon, ESL, LS002294) for 30 min at 37 °C. To stop the digestion, fetal bovine serum (FBS, Invitrogen, 10082-139) was added to a final concentration of 20%. Tissues were triturated until homogenous and filtered through a 70 µm cell strainer (Falcon, 352350) on ice in a 4 °C cold room and transferred to a 2 mL tube. The sample was then centrifuged at 1,537 rcf for 1 min; then 1 mL red blood cell lysis buffer (15 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA, pH 8.0) was added and incubated for 3 min before centrifugation again at 1,537 rcf. The red blood cell lysis buffer incubation was repeated to remove residual red blood cells. Liebovitz+10% FBS was used to wash and resuspend samples, which were then filtrated through a 35 mm cell strainer into a 5 mL glass tube and had SYTOX Blue (1:1000, Invitrogen, S34857) added. After refiltering, samples were for ATAC-seq were sorted for GFP positive cells from a RosaSun1GFP reporter activated by Wnt3aCre, Rtkn2CreER, or SftpcCreER on a BD FACSAria IIIu cell sorter with a 70 mm nozzle. Samples for scRNA-seq or scATAC-seq were stained with CD45-PE/Cy7 (BioLegend, 103114), ECAD-PE (BioLegend, 147304), and ICAM2-A647 (Invitrogen, A15452) antibodies (1:250 dilutions for all antibodies) for 30 min followed by a wash and being resuspended with Liebovitz meda+10% FBS and addition of SYTOX Blue. Samples were then filtered through a 35 mm cell strainer into a 5 mL glass tube again and sorted by a BD FACSAria Fusion sorter or a with a 70 mm nozzle.
FACS-purified lung epithelial, immune, endothelial, and mesenchymal cells were processed through the Chromium Single Cell Gene Expression Solution Platform (10X Genomics) using the Chromium Single Cell 3' Library and Gel Bead Kit in accordance with the manufacturer’s user guide (v2, rev D).
The libraries were sequenced on an Illumina NextSeq500 using a 26X124 sequencing run format with 8 bp index (Read1).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
control lungs
Aggregate12files_control_scRNAseq_barcodes.tsv
Aggregate12files_control_scRNAseq_genes.tsv
Aggregate12files_control_scRNAseq_matrix.mtx
Aggregate12files_control_scRNAseq_cloupe_processed.cloupe
E16-5-control
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| Data processing |
Chromium single-cell RNA-seq output was processed with Cell Ranger using “cellranger count” and “cellranger aggr”.
Further analysis was carried out using Loupe Cell Browser (10x Genomics) or an R package Seurat
Trajectory analysis was carried out using Monocle 2.8
Genome_build: mm10
Supplementary_files_format_and_content: tsv and mtx files can be opened with Seurat (R package) and analyzed. Cloupe file can be opened with Loupe cell browser to visualize the data.
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| Submission date |
Sep 18, 2020 |
| Last update date |
Mar 08, 2021 |
| Contact name |
Danielle R. Little |
| E-mail(s) |
Danielle.Little@stjude.org
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| Phone |
9015953487
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| Organization name |
St. Jude Children's Research Hospital
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| Department |
Developmental Neurobiology
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| Lab |
Michael Dyer
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| Street address |
262 Danny Thomas Pl Rm D2031
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| City |
Memphis |
| State/province |
Tennessee |
| ZIP/Postal code |
38105 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE158192 |
Differential chromatin binding of the lung lineage transcription factor NKX2-1 resolves opposing murine alveolar cell fates in vivo [scRNA-Seq] |
| GSE158205 |
Differential chromatin binding of the lung lineage transcription factor NKX2-1 resolves opposing murine alveolar cell fates in vivo |
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| Relations |
| BioSample |
SAMN16205824 |
| SRA |
SRX9148773 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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