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| Status |
Public on Apr 16, 2024 |
| Title |
E16.5_WT_lung |
| Sample type |
SRA |
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| Source name |
lung
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| Organism |
Mus musculus |
| Characteristics |
tissue: lung
cell type: epithelial, mesenchymal, endothelial and immune
genotype: WT
age: Embryonic day 16.5
treatment: no treatment
Sex: pooled male and female
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| Extracted molecule |
genomic DNA |
| 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 FACS-purified lung epithelial, immune, endothelial, and mesenchymal c, were processed using the 3′ Library and Gel Bead Kit following the manufacturer’s users guide (v2 rev D) on the Chromium Single-Cell Gene Expression Solution Platform (10X Genomics).
Libraries were then sequenced using Illumina Novaseq6000 with a 50-paired-end format with index1 for 8 cycles and index2 for 16 cycles.
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| Library strategy |
ATAC-seq |
| Library source |
genomic single cell |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
wild type mouse lung
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| Data processing |
Chromium single-cell ATAC-seq output was processed with Cell Ranger using “cellranger count” and “cellranger aggr”.
Bams were filtered using Picard and Samtools with the commands MarkDuplicates and –f 3 –F 4 –F 256 –F 1024 –F 2048 –q 30
Barcodes were extracted from processed cell clusters in Seurat and Signac and used with Sinto to partition the cellranger output bam file into smaller cell type bams
Further analysis can be achieved using the Cell Ranger ATAC’s functions “cellranger-atac count”. Downstream analysis was performed using Seurat R package (4.3) Signac (1.9)
Peaks were then called using Macs on the -braod setting whereupon peaks were used for downstream analysis.
Assembly: mm10
Supplementary files format and content: Rds files can be opened with Signac (R package) and analyzed
Supplementary files format and content: bw files can be opened with igv genomic browser
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| Submission date |
Apr 16, 2024 |
| Last update date |
Apr 18, 2024 |
| Contact name |
Dalia Hassan |
| E-mail(s) |
[email protected]
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| Phone |
3467146254
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| Organization name |
MD Anderson Cancer Center
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| Department |
Pulmonary Medicine
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| Lab |
Jichao Chen
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| Street address |
6565 MD Anderson Blvd, Z9.4040, Z9.4040
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| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE264098 |
CEBPA restricts alveolar type 2 cell plasticity during development and injury-repair [scATAC-seq] |
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| Relations |
| BioSample |
SAMN41008088 |
| SRA |
SRX24301361 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8210407_E16.5_wtSox9progenitors_ATAC_s1.broadPeak.gz |
2.5 Mb |
(ftp)(http) |
BROADPEAK |
| GSM8210407_E16.5_wtSox9progenitors_ATAC_s1.bw |
244.0 Mb |
(ftp)(http) |
BW |
| GSM8210407_E16.5_wtSox9progenitors_ATAC_s2.broadPeak.gz |
2.5 Mb |
(ftp)(http) |
BROADPEAK |
| GSM8210407_E16.5_wtSox9progenitors_ATAC_s2.bw |
287.3 Mb |
(ftp)(http) |
BW |
| GSM8210407_E165_WT_scATAC.rds.gz |
1.4 Gb |
(ftp)(http) |
RDS |
SRA Run Selector |
| Raw data are available in SRA |
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