|
| Status |
Public on Mar 31, 2013 |
| Title |
FoxA1 ChIP-seq in Mitotic Cells 1 |
| Sample type |
SRA |
| |
|
| Source name |
HuH7 cells, mitotic block and release
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: HuH7
cycling state: mitotic
antibody: FoxA1
vendor/catalog/lot: Abcam ab23738 (lot #737921)
|
| Treatment protocol |
Cells were blocked in S phase by addition of fresh medium containing 2 mM thymidine (Sigma; St. Louis, MO; T1895). After 24 hours, cells were washed three times with PBS and released into medium containing 0.06 µg/mL nocodazole (Sigma; M1404). After 18 h, about 94% of the cells were blocked in metaphase (Fig. S5a) and used for imaging or ChIP. For mitotic block-release studies, arrested cells were either: 1) harvested by gentle shaking and replated into fresh medium, or 2) washed 3 times with PBS (2 minutes incubation at room temperature per wash) and fresh medium was added to the cells.
|
| Growth protocol |
HuH7 cells were plated in fresh DMEM High Glucose with L-glutamine (Invitrogen; Carlsbad, CA; 11965) containing 10% FBS (HyClone, Logan, UT) and 1% penicillin-streptomycin (Invitrogen) and grown overnight to 75% confluence.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Triplicate immunoprecipitation samples were adjusted to 335 µg and then divided into two aliquots. The incubation with FoxA1 antibodies were performed with 18 µg. ChIP products were used to generate libraries using a ChIP-Seq sample preparation kit (illumina IP-102-1001, San Diego CA). Amplicons between 100 and 200 bp were validated on a Bioanalyzer (Agilent Technologies 2100, Santa Clara CA) before being used for sequencing on an Illumina Sequencer.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
DNA immunoprecipitated by FoxA1
|
| Data processing |
ELAND was used to perform base-calls (default parameters)
Quality control and alignment was performed using ELAND (default parameters)
MACS was used to detect peaks in FoxA1 binding (MFOLD=16, FDR=0.05 for mitotic and 0.0005 for asynchronous cells after pooling replicates
Track views were generated by pooling replicates, then binning all aligned tags into 25bp bins and normalizing by the total number of bases sequenced, then subtracting input
Genome_build: hg18 (NCBI 36)
Supplementary_files_format_and_content: BED6 files are included for called peaks
|
| |
|
| Submission date |
Jul 10, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Kenneth S. Zaret |
| E-mail(s) |
zaret@pennmedicine.upenn.edu
|
| Phone |
2155735813
|
| Organization name |
University of Pennsylvania School of Medicine
|
| Department |
Cell and Developmental Biology
|
| Lab |
Zaret lab
|
| Street address |
9-132, SCTR, 3400 Civic Center Boulevard
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104-5157 |
| Country |
USA |
| |
|
| Platform ID |
GPL10999 |
| Series (2) |
| GSE39241 |
FoxA1 binding in asynchronous and mitotic HUH7 cells |
| GSE39243 |
Expression and FoxA1 binding in asynchronous and mitotic HUH7 cells |
|
| Relations |
| SRA |
SRX159092 |
| BioSample |
SAMN01086977 |
