|
| Status |
Public on Nov 08, 2022 |
| Title |
NMF repetition 1 co-cultured with 4T1 cells for 3 days |
| Sample type |
SRA |
| |
|
| Source name |
Normal mammary fibroblasts
|
| Organism |
Mus musculus |
| Characteristics |
strain: BALB/c
tissue: Mammary fat pad
cell type: Normal mammary fibroblasts
selection marker: GFP- PDPN+
treatment: co-cultured with 4T1 cells for 3 days
mouse age: 12 weeks
|
| Treatment protocol |
3*10^5 Normal mammary fibroblasts were plated in 6 wells in DMEM supplemented with 10% FBS and pen/strep. 24h later 1*10^5 4T1-GFP cells were seeded on top of the NMF. 3 or 5 days later cells were collected into single-cell suspensions.
|
| Growth protocol |
Fresh mammary fat pad tissue was extracted from 12-week-old female BALB/c mice. The tissue was minced and digested using gentleMACS dissociation buffer (DMEM with 1mg/ml collagenase II , 1mg/ml collagenase IV, and 0.01mg/ml deoxyribonuclease. To stop the reaction, 20ml of DMEM with 10% FBS and 1% Penicillin- Streptomycin were added to the dissociation buffer followed by filtration with a 70μm strainer. Red blood cells (RBC) were removed by RBC lysis buffer. For the co-culture assay, the cells were cultured on a collagen layer for 1 week of recovery
|
| Extracted molecule |
total RNA |
| Extraction protocol |
cell suspensions were stained with anti-PDPN antibodies. NMF were isolated by FACS sorting using negative selection for GFP and positive selection for PDPN
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 |
| GSE213071 |
Global DNA methylation analysis of cancer-associated fibroblasts reveals extensive epigenetic rewiring linked with RUNX1 upregulation in breast cancer stroma [co-culture] |
|
| Relations |
| BioSample |
SAMN30788995 |
| SRA |
SRX17520816 |
