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| Status |
Public on Apr 13, 2024 |
| Title |
Cracd WT, scRNAseq |
| Sample type |
SRA |
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| Source name |
Lung
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| Organism |
Mus musculus |
| Characteristics |
tissue: Lung
cell type: Epithelial cell
genotype: Cracd WT
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| Extracted molecule |
total RNA |
| Extraction protocol |
Lungs were harvested from euthanized mice (Cracd WT or Cracd KO) after perfusing 10 ml of cold phosphate-buffered saline (PBS) into the right ventricle. The lung was digested in Leibovitz’s medium (Invitrogen) with 2 mg/mL Collagenase Type I (Worthington), 2 mg/mL Elastase (Worthington), and 2 mg/mL DNase I (Worthington) at 37 °C for 45 min. The tissue was triturated with a pipet every 15 min of digestion until homogenous. The digestion was stopped with FBS (Invitrogen) to a final concentration of 20%. The cells were filtered with a 70 μm cell strainer (Falcon) and spun down at 5,000 r/min for 1 min. The cell pellet was resuspended in red blood cell lysing buffer (Sigma) for 3 min, spun down at 5,000 r/min for 1 min, and washed with 1 mL ice-cold Leibovitz’s medium with 10% FBS. In single-cell RNA sequencing (scRNA-seq), digested lung cells were resuspended in 400 μl of buffer with 5 μl of anti-CD31-FITC (BD Biosciences, CA, USA), 5 μl of anti-CD45-APC (BD Biosciences), and 5 μl of anti-CD326 (EpCAM)-PE-Cy7 (BD Biosciences) and incubated for 30 min at 4 C. Cells were then washed twice, followed by sorting of the epithelial cells (EpCAM+ / CD31- / CD45-) by fluorescence-activated cell sorting at the Cytometry and Cell Sorting Core at BCM.
Single-cell Gene Expression Library was prepared according to the guideline for the Chromium Single Cell Gene Expression 3v3.1 kit (10X Genomics). Briefly, single cells, reverse transcription (RT) reagents, Gel Beads containing barcoded oligonucleotides, and oil were loaded on a Chromium controller (10 Genomics) to generate single-cell GEMS (Gel Beads-In-Emulsions), where full-length cDNA was synthesized and barcoded for each single cell. Subsequently, the GEMS were broken and cDNAs from each single cell were pooled, followed by cleanup using Dynabeads MyOne Silane Beads and cDNA amplification by PCR. The amplified product was then fragmented to optimal size before end-repair, A-tailing, and adaptor ligation. The final library was generated by amplification. The library was performed at the Single Cell Genomics Core at BCM.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The Cell Ranger was used for demultiplexing, barcoded processing, and gene counting. The loom files were generated using velocyto package 32. The R package Seurat33 and Python package Scanpy34 were used for pre-processing and clustering of scRNA-seq data with the loom files. UMAP was used for dimensional reduction, and cells were clustered in Seurat or Scanpy. Datasets were pre-processed, normalized separately. Each dataset was normalized separately and clustered by the “Leiden” algorithm 35. Cracd WT and Cracd KO datasets were combined using “ingest” function in Scanpy. Scanpy was used to concatenate the Cracd WT vs. KO dataset and preSC Cracd WT vs. KO samples. Cells with more than 7000 counts reads were removed. Gene expression for each cell was normalized and log-transformed. The percentages of mitochondrial reads were regressed before scaling the data. Dimensionality reduction and Leiden clustering (resolution 0.5 ~ 1) was carried out, and cell lineages were annotated based on algorithmically defined marker gene expression for each cluster (sc.tl.rank_genes_groups, method=‘wilcoxon’). The list of differentially expressed genes (DEGs) in Cracd WT and Cracd KO was generated by comparing Cracd KO vs. Cracd WT (sc.tl.rank_genes_groups, groups=[' Cracd KO '], reference= ‘Cracd WT’, method='wilcoxon').
Assembly: mm10
Supplementary files format and content: h5ad anndata
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| Submission date |
Apr 18, 2023 |
| Last update date |
Apr 13, 2024 |
| Contact name |
Bongjun Kim |
| E-mail(s) |
arrice86@gmail.com
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| Organization name |
MD Anderson Cancer Center
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| Department |
Experimental Radiation Oncology
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| Lab |
Jae-Il Park Lab
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| Street address |
6565 MD Anderson BLVD
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| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE229982 |
Gene expression profiles at single cell level of lung epithelial cells from the Cracd WT and Cracd KO mice |
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| Relations |
| BioSample |
SAMN34234324 |
| SRA |
SRX20001801 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7182335_CracdWT.h5ad.gz |
298.2 Mb |
(ftp)(http) |
H5AD |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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