|
| Status |
Public on Jan 17, 2020 |
| Title |
PdgfraEGFP Single Cell RNA-seq |
| Sample type |
SRA |
| |
|
| Source name |
PDGFRAEGFP+ cells from small intestinal mesenchyme
|
| Organism |
Mus musculus |
| Characteristics |
cell type: primary cells
cell subtype: Mixed telocyte and PDGFRAlo stromal cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were isolated from entire small intestinal tissues, muscle stripped, epithelium denuded via 20 minutes in warm 10 mM EDTA/HBSS (at 37degrees C), and tissue was further minced by fine scalpels. Digestion of tissue was performed using 1.5mg/ml collagenase II (Worthington) in 10%FBS/DMEM at 37 degrees C. Supernatants were collected every 20 minutes for one hour, and washed twice to remove debris. Antibody-labelling was performed prior to FACS. GFP cells were FACS sorted at a ratio of 2:1 GFPlo to GFPhi. A manually estimated 8700 cells were loaded on a 10X Chromium Controller (10X Genomics) for targeting 5000 cells using the single cell 3' V2 assay. Reverse-transcription, cDNA amplification and library prep were compelted according to manufacturer's protocol. Sequencing of library was performed using a HiSeq4000 Illumina System.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Cell Ranger (2.1.1, 10X Genomics) was used to generate UMI matrix. Data was analyzed using the Suerat package (ver. 2.3.3) in R. Cells with atleast 1500 detected genes and genes expressing in at least 100 single cells were kept. This resulted in 9,334 detectable genes in 2,894 singel cells. The data was then normalized and log-transformed using the "LogNormalize" function in Seurat. We additionally filtered out cells with high mitochondrial percentage (>3%) which resulted in 2,595 single cells that were used for the rest of the analysis.
All bulk RNAseq data was analyzed using the VIPER pipeline
It was aligned to the mouse reference genome Mm10 using the STAR aligner
Genome_build: mm10
Supplementary_files_format_and_content: Rawgenescounts file was generated using the VIPER pipeline, aligned to the mm10 reference genome using STAR aligner, which also produced gene counts.
|
| |
|
| Submission date |
May 03, 2019 |
| Last update date |
Feb 11, 2020 |
| Contact name |
Ramesh Shivdasani |
| E-mail(s) |
ramesh_shivdasani@dfci.harvard.edu
|
| Organization name |
Dana Farber Cancer Institute
|
| Department |
Medical Oncology
|
| Lab |
Shivdasani
|
| Street address |
450 Brookline Ave. Dana 720D
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL21103 |
| Series (1) |
| GSE130681 |
Distinct mesenchymal cell populations generate the essential intestinal BMP signaling gradient |
|
| Relations |
| BioSample |
SAMN11569564 |
| SRA |
SRX5785325 |
