|
| Status |
Public on Mar 28, 2023 |
| Title |
Tgfb_PFI3 |
| Sample type |
SRA |
| |
|
| Source name |
Aorta and carotid artery
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Aorta and carotid artery
cell line: AdvSca1-SM cells
cell type: Resident vascular progenitor
treatment: Tgfb and PFI-3
|
| Treatment protocol |
On the day of treatment, growth-arrested AdvSca1-SM cells were switched to DMEM media supplemented with 0.1% FBS and 1x penicillin/streptomycin and treated with either 5ng/mL TGF-B by adding a 5ug/mL stock solution of TGFB at a 1:1000 dilution (diluted in serum-free DMEM) to the cells, or treated with 5ng/mL TGF-B + 50uM PFI-3. For cells treated with PFI-3, a 50mM stock was applied at a 1:1000 dilution in DMSO. All cells received 0.1% DMSO as vehicle control. Cells were incubated for 72 hours.
|
| Growth protocol |
AdvSca1-SM cells were maintained in alpha-MEM GlutaMAX media supplemented with 10% mesenchymal stem cell-qualified fetal bovine serum (FBS), 1ng/mL murine basic fibroblast growth factor, 5ng/mL murine epidermal growth factor, and 1x penicillin/streptomycin in humidified atmosphere with 5% CO2 at 37C. Prior to treatment, AdvSca1-SM cells were maintained in alpha-MEM GlutaMAX media supplemented with 0.1% mesenchymal stem cell-qualified fetal bovine serum (FBS), 1ng/mL murine basic fibroblast growth factor, 5ng/mL murine epidermal growth factor, and 1x penicillin/streptomycin for 24hrs to growth arrest cells
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
On the day of extract, cells were trypsinized with 0.25% trypsin and washed three times in wash buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine supplemented with protease inhibitor cocktail. The cells were then adsorbed to Concanavalin A beads for 20 minutes at room temperature. Cells were then permeabilized with Antibody Buffer containing 0.1% digitonin and 2mM EDTA. Permeabilization was validated by Trypan Blue. Samples were incubated with 0.5μg of anti-Brg1, anti-H3K4me3, or IgG antibody overnight at 4°C. The following day, cells were washed twice in wash buffer plus 0.1% digitonin and incubated with pAG-micrococcal nuclease (pAG-MNase) for 20 minutes at room temperature and then cells were incubated with 1mM CaCl2 for 2 hours at 4°C to activate pAG-MNase digestion of chromatin fragments under target. The digest reaction was quenched using EpiCypher Stop Buffer and samples were incubated at 37°C for 30 minutes to liberate digested DNA. The enriched DNA was purified using the EpiCypher DNA column purification kit
Libraries were prepared with the Tecan/Nugen Ultralow DNA Input Kit for end repair and adapter ligation, Paired End 150 cycle 2x150 with 14 cycles of amplification and library distribution was evaluated using TapeStation.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
CUT&RUN reads were quality assessed by FastQC (v0.11.8), MultiQC (v1.8), FastQ Screen (v0.14.0) and quality filtered with BBDuk (v38.87).
Sequencing reads were aligned to the mm10 mouse using Bowtie2 (v2.3.4.3).
Samtools (v.1.11) was used to select the uniquely mapped reads (samtools view -b - q 30) and sort the bam files.
PCR duplicates were removed using Picard MarkDuplicates (v2.21.1) tool.
The normalization ratio of each sample was calculated by dividing the total number of mapped reads mapping to the murine genome of each sample by the total number of mapped reads mapping to the murine genome of the sample with the lowest number of reads mapping to the murine genome. Using the normalization ratio, random sub-sampling of the reads was performed using samtools view -s.
Bedtools genomecov was used to create bedgraph files from the bam files.
Bigwig files were generated with DeepTools (v3.5.1) and visualized with IGV (v2.13.2).
Peaks were called using MACS2 (v2.1.2) with default parameters.
Assembly: mm10
Supplementary files format and content: bigwig files
|
| |
|
| Submission date |
Aug 17, 2022 |
| Last update date |
Mar 28, 2023 |
| Contact name |
Mary C.M. Weiser-Evans |
| E-mail(s) |
Mary.weiser@ucdenver.edu
|
| Phone |
303-724-4846
|
| Organization name |
University of Colorado Anschutz Medical Campus
|
| Department |
Renal Diseases & Hypertension
|
| Lab |
Dr. Mary C.M. Weiser-Evans
|
| Street address |
12700 East 19th Avenue, C281
|
| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE211422 |
The chromatin remodeler Brg1 directs smooth muscle-derived adventitial progenitor-to-myofibroblast differentiation and in situ vascular fibrosis [CUT&RUN] |
| GSE211423 |
The chromatin remodeler Brg1 directs smooth muscle-derived adventitial progenitor-to-myofibroblast differentiation and in situ vascular fibrosis. |
|
| Relations |
| BioSample |
SAMN30360564 |
| SRA |
SRX17115033 |
