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| Status |
Public on Nov 08, 2022 |
| Title |
pCAF Biological repetition 1 |
| Sample type |
SRA |
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| Source name |
PDPN+ CAFs
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| Organism |
Mus musculus |
| Characteristics |
strain: BALB/c
tissue: Breast tumor
cell type: Non-immune stromal cells
selection marker: Ter119- CD45- EpCAM- PDPN+
treatment: Orthotopic breast injection of 4T1-luc cells
mouse age: 8 weeks
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| Treatment protocol |
8 weeks old BALB/c females were injected under anaesthesia with 100,000 4T1-luc 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.
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| Growth protocol |
8 to 13 weeks-old mice housed at the Weizmann Institute animal facility under specific pathogen-free conditions.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Normal mammary fat pad: Tissue was minced using scissors and placed and disrupted for 25 min at 37oC in a gentleMACS C tube using gentleMACS dissociator, in the presence of enzymatic digestion solution containing 1 mg ml-1 collagenase II, 1 mg ml-1collagenase IV and 70 unit ml-1 DNase. 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 1640 for 20 min at 37oC, with pipetting every 3 min.
DNA from NMFs and pCAFs was processed for tWGBS as described in Weichenhan et al. 2018, Briefly, we started with 25ng DNA for NMF, 8.5 ng for pCAFs. For the tagmentation reaction, we added 2µl of EZ-Tn5TMTransposase (Lucigen, cat# TNP92110), 1µl Unmethylated phage λ DNA and 5µl tagmentation buffer (20 mM Tris(hydroxymethyl)aminomethane, 10 mM MgCl2, 20% (vol/vol) dimethylformamide). DNA was purified using 46µl AMPure beads (Agencourt AMPure XP beads) following the manufacturer’s protocol. Next, we performed an Oligo Replacement/Gap Repair reaction using 2μl of dNTP mix. The bisulfite treatments were achieved by EZ DNA Methylation™ Kit (ZYMO Research) following the commercial kit protocol. The samples were divided into 4 technical replicates with different barcodes. We performed a qPCR reaction to amplify the libraries, followed by AMPure bead purification. The samples were eluted in 10μl of EB buffer (Tris-HCL pH8), library concentration was quantified with Qubit, and average fragment size was detected by Bioanalyzer. Libraries were sequenced using paired-end sequencing, 125 bps, on one lane of a HiSeq2000 v4 sequencer (Illumina, San Diego, CA, USA) per 4 technical replicates.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Raw reads were trimmed using Trimmomatic v0.36 and aligned against the mouse reference genome (mm10) utilizing bwa mem v0.7.8, invoking the parameter "-T 0". Alignment duplicates were marked by Picard MarkDuplicates (http://broadinstitute.github.io/picard) v1.125. Reads with coverage > 2 were sorted, and PCR duplicates were removed. Methylation calling was executed with MethylDackel (https://github.com/dpryan79/MethylDackel) v0.3.0. According to the M-bias plots, the first five base pairs at the two ends of the reads were excluded from methylation calling.
Genome_build: mm10
Supplementary_files_format_and_content: tab delimited text files include methylation calls and coverages per sample per CpG
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| Submission date |
Jan 12, 2022 |
| Last update date |
Nov 08, 2022 |
| Contact name |
Coral Halperin |
| E-mail(s) |
coral.halperin@weizmann.ac.il
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| Organization name |
Weizmann institute of sciences
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| Lab |
Scherz-Shouval
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| Street address |
Herzel 234
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| City |
rehovot |
| ZIP/Postal code |
76100 |
| Country |
Israel |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE193518 |
Global DNA methylation analysis of cancer-associated fibroblasts reveals extensive epigenetic rewiring linked with RUNX1 upregulation in breast cancer stroma [WGBS] |
| GSE195865 |
Global DNA methylation analysis of cancer-associated fibroblasts reveals extensive epigenetic rewiring linked with RUNX1 upregulation in breast cancer stroma |
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| Relations |
| BioSample |
SAMN24892441 |
| SRA |
SRX13747607 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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