|
| Status |
Public on Jan 01, 2019 |
| Title |
RASMC_WCE_PDGF_2H_NEW |
| Sample type |
SRA |
| |
|
| Source name |
Spike in normalized ChIP-Seq for H3k27ac in RASMC, 2hr after PDGF treatment
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell line: RASMC
tissue: Rat aortic smooth muscle cell
input: NONE
barcode: AGTCAA
antibody: None
antibody maker: None
antibody catalog number: None
|
| Treatment protocol |
For PDGF treatment, cells were treated with PDGF-BB (25ng/ml) for 24 hours
|
| Growth protocol |
Rat aortic smooth muscle cells (RASMCs), obtained from Lonza, were cultured in DMEM:F12 media supplemented with 10% v/v FBS, 100 U/ml penicillin-streptomycin, and 2 mM L-glutamine at 37 °C in a humidified atmosphere of 95% air and 5% CO2 incubator. Cells were starved in serum free media for 24 hours before starting any treatment.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed as described previously (PMID: 27425608). Briefly, cells were fixed with 1% formaldehyde for 10min, quenched with 0.125M glycine for 5min and washed with ice cold PBS containing protease inhibitor. Cell pellets were snap frozen in liquid nitrogen and saved in -80oC. Cells were lysed in lysis buffer as described here PMID: 27425608 and chromatin was sheared using Diagenode Bioruptor plus (16 min, 30 sec on/30 sec off, setting high). Immunoprecipitation was performed using 25 million cells for each H3k27Ac (Abcam, ab4729, 5µg per IP) and RNA Pol II (Biolegend 920102 Clone 8WG16, 5µl per IP). 75 million cells and 5µg antibody was used for Brd4 IP (Bethyl A301-985A). IP Samples were reverse cross linked and DNA was purified using phenol:chloroform:isoamyl alcohol. Purified DNA samples were sent to WIGTC for NGS library preparation and sequencing.
TruSeq ChIP library preparation kit was used according to manufacturer's instructions (https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-chip.html)
ChIP-Rx (PMID:25437568) with single end 40bp sequencing
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Sequenced reads were aligned to a custom reference bowtie2 index combining the RN6 and DM6 genome builds
ChIP_RX scaling factors were determined as in Orlando et al., 2014, "Quantitative ChIP-Seq normalization reveals global modulation of the epigenome", PMID 25437568
Peaks were called using MACS1.4.2 with p-val =1e-9 and background datasets as described in characteristic: input
Genome_build: rn6:dm6
Supplementary_files_format_and_content: Processed ChIP-Rx datasets are provided in a cell count normalized scaled and unscaled wig format. File RN6_RASMC_NEW_CHIPRX_SCALE_FACTORS.txt contains ChIP-Rx scale factors for each dataset
|
| |
|
| Submission date |
Mar 12, 2018 |
| Last update date |
Jan 01, 2019 |
| Contact name |
Charles Yang Lin |
| E-mail(s) |
charles.y.lin@bcm.edu
|
| Phone |
6172764723
|
| Organization name |
Baylor College of Medicine
|
| Department |
Molecular and Human Genetics
|
| Street address |
1 Baylor Plaza
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL18694 |
| Series (2) |
| GSE111710 |
Dynamic rewiring of transcription factor networks during smooth muscle cell phenotypic modulation (ChIP-Rx data sets) |
| GSE111715 |
Dynamic rewiring of transcription factor networks during smooth muscle cell phenotypic modulation |
|
| Relations |
| BioSample |
SAMN08687459 |
| SRA |
SRX3782454 |
