|
| Status |
Public on Nov 08, 2022 |
| Title |
Ly6C+ pCAF repetition 3 |
| Sample type |
SRA |
| |
|
| Source name |
Ly6c+ PDPN+ CAFs
|
| Organism |
Mus musculus |
| Characteristics |
strain: BALB/c
tissue: Breast tumor
cell type: Non-immune stromal cells
selection marker: Ter119- CD45- EpCAM- PDPN+ LY6C+
treatment: Orthotopic breast injection of 4T1-luc cells
mouse age: 8 weeks
|
| Treatment protocol |
8 week old BALB/c females were injected under anaesthesia with 100,000 4T1-GFP cells suspended in PBS, directly into the lower left mammary fat pad. To obtain CAFs from primary tumors, mice were sacrificed and tumors were immediately excised post mortem.
|
| Growth protocol |
8 week-old mice housed at the Weizmann Institute animal facility under specific pathogen-free conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Primary tumor: Tissue was minced using scissors and treated with enzymatic digestion solution containing 3 mg ml-1 collagenase A and 70 unit ml-1 DNase in RPMI in the gentleMACS dissociator. The digested cell suspension was strained through a 70µm cell strainer to obtain a single-cell suspension. To isolate CAFs, we performed negative selection against cancer cells (EpCAM) and immune cells (CD45) the Ly6c pCAFs pCAF were selected based on PI−, Ter119−, CD45−, EpCAM−, PDPN+ , LY6C+/-
3' end mRNA libraries were prepared for sequencing using a method developed at Dr. Ido Amit's lab, Weizmann Institute of Science
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Illumina bcl2fastq software used for base calling.
Sample barcodes were extracted from read 2 and concatenated to the fastq header of read 1. Barcode is of size 7 followed by UMI of size 8
aligment: STAR v2.4.2a parameters: –alignEndsType EndToEnd, –outFilterMismatchNoverLmax 0.05, –twopassMode Basic, –alignSoftClipAtReferenceEnds No
We used the 3’ end (1000bp) of the transcripts for counting the number of reads per gene. Counting (UMI counts) was done after marking duplicates (in-house script) using HTSeq-count (DOI: 10.1093/bioinformatics/btu638) in union mode.
Further analysis was done for genes having minimum 5 reads in at least one sample
Assembly: mm10
Supplementary files format and content: tab delimited text files include mRNA molecule normalized counts values for each sample
|
| |
|
| Submission date |
Sep 10, 2022 |
| Last update date |
Nov 08, 2022 |
| Contact name |
Coral Halperin |
| E-mail(s) |
coral.halperin@weizmann.ac.il
|
| Organization name |
Weizmann institute of sciences
|
| Lab |
Scherz-Shouval
|
| Street address |
Herzel 234
|
| City |
rehovot |
| ZIP/Postal code |
76100 |
| Country |
Israel |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE195865 |
Global DNA methylation analysis of cancer-associated fibroblasts reveals extensive epigenetic rewiring linked with RUNX1 upregulation in breast cancer stroma |
| GSE213074 |
Global DNA methylation analysis of cancer-associated fibroblasts reveals extensive epigenetic rewiring linked with RUNX1 upregulation in breast cancer stroma [ly6c] |
|
| Relations |
| BioSample |
SAMN30789032 |
| SRA |
SRX17520840 |
