|
| Status |
Public on Oct 18, 2013 |
| Title |
Differentiating_H3K4me3_ChIPSeq |
| Sample type |
SRA |
| |
|
| Source name |
H9 hESC
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: RA-mediated differentiating hESC
chip antibody: H3K4me3
antibody vendor: Milipore
|
| Treatment protocol |
For differentiating hESCs cultured on mTeSR1, 1 μM Retinoic Acid (RA) was added to homemade MEF-CM (Conditioned Media, without additional FGF). CM media was prepared in our facility by culturing γ-irradiated MEFs in complete hESC culture media for 24 hours, and collected every day, filtered and freezed at -20 degrees. FGF was added to CM media before use to a final concentration of 10 ng/ml to culture the cells grown on Matrigel under pluripotent conditions.
|
| Growth protocol |
Human Embryonic Stem (WA09) Cells were obtained from National Stem Cell Bank (Madison, WI) and cultured according to the protocol from WiCell Research Insitute. Briefly, WA09 cells were maintained in hESC culture medium on γ-irradiated Mouse Embryonic Fibroblasts (MEFs) prepared using WiCell instructions. hESCs ranging from passage number 32-38 were used for all of our experiments. hESC complete culture medium is composed of DMEM/F12 supplemented with 20% knockout serum replacement, 1 mM L-glutamine, 1% nonessential amino acids, 4 ng/ml human FGF2 (all from Invitrogen), and 0.1 mM 2-mercaptoethanol (Sigma). The medium was changed daily, and cells were passaged every 4–6 days with 1 mg/ml Collagen IV (Invitrogen). For differentiation studies hESCs were cultured in differentiation media (hESC media without FGF) containing 1 μM Retinoic Acid (RA) with fresh medium change daily. hESCs were also maintained as feeder-free cultures on hESC qualified Matrigel (BD Biosciences) in mTeSR1 media (Stem Cell Technologies) and CM (MEF conditioned media). Passage 32 hESCs were grown on mTeSR1 media for 5 passages. hESCs were cultured on Matrigel (BD Biosciences) following manufacturer’s instructions, received fresh mTeSR1 media everyday and cells were passaged every 4-6 days with 1 mg/ml Dispase (Stem Cell Technologies).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Sequence reads were obtained and mapped to the February 2006 human reference sequence (hg18) using the Illumina Genome Analyzer Pipeline using Illumina’s ELAND software.
Enriched regions for each condition were normalized to input DNA and detected with MACS (Zhang et al., 2008) with a cut-off P < 10-8 for Damage and Differentiation P< 10-10 for Untreated.
Genome_build: hg18
Supplementary_files_format_and_content: peak and aligned bed files
|
| |
|
| Submission date |
Aug 06, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Kadir Caner Akdemir |
| E-mail(s) |
kcakedemir@mdanderson.org
|
| Organization name |
MD Anderson Cancer Center
|
| Street address |
1515 Holcombe Blvd
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE39912 |
Genome Wide Profiling of p53 Response to Differentiation or DNA Damage of Human Embryonic Stem Cells |
|
| Relations |
| SRA |
SRX189255 |
| BioSample |
SAMN01728984 |
