|
| Status |
Public on Mar 01, 2013 |
| Title |
GSK_input |
| Sample type |
SRA |
| |
|
| Source name |
mouse embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell line: Ctnnb1-Biotin-3xFLAG knock-in V6.5 ESC line
chip-antibody: none
treatment: CHIR99021 (3uM)
|
| Treatment protocol |
Cells were treated for 16 hours with CHIR99021 (3 μM)
|
| Growth protocol |
All ESCs were maintained in complete ES media (15% fetal bovine serum, 0.1 mM non-essential amino acids, 0.1 mM b-mercaptoethanol, 2 mM L-glutamine, 10^3 units/ml LIF, and 1X nucleotide mix in DMEM high glucose) with feeder cells isolated from DR4 mice
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After treated with CHIR99021 (3 mM ) for 16 hours, Ctnnb1-BioFLneo;BirA and BirA ESC were collected with feeder cells using 0.25% trypsin-EDTA. After leaving cells on gelatinized plates for an hour, we collected floating cells as an ESC-enriched fraction. 10,000,000 cells were used per ChIP. Chromatin was sonicated by 6 sessions of 30 pulses (1 sec on and 1 sec off) at 50% amplitude using the Branson sonifier 250D. Isolation of chromatin and FLAG-ChIP were performed as described (Development. 2007;134:1977-1989). For Biotin-ChIP, sonicated chromatins were first incubated with protein A dynabeads (Invitrogen, 100-01D) at 4 °C for an hour. Dynabeads MyOne Streptavidin T1 (Invitrogen, 656.01) was incubated with 2% Gelatin from cold water fish skin (Sigma, G7041) in PBS at 4 °C for an hour prior to use. Precleared chromatins were incubated with 60 mL of Dynabeads MyOne Streptavidin T1 overnight with rotation at 4 °C. Beads were washed with 2% SDS, 10 mM Tris, and 0.5 M EDTA five times, and with TE once. ChIP DNA was eluted by the overnight incubation of beads in 10 mM Tris, 1 mM EDTA, 1% SDS, and 0.5 M NaCl at 65 °C. DNA was purified as described (Development. 2007;134:1977-1989).
ChIP-seq libraries were constructed using ChIP-seq DNA Sample Prep Kit (Illumina, IP-102-1001) according to manufacturer’s instruction.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Data processing |
Base calling was performed using Illumina pipeline
The firsty 25 bp of sequence reads were mapped to unmasked mouse genome using either seqmap or eland allowing up to two mismatches
Peak calling was performed using MACS1.3 with p-value cutoff 1e-5.
Genome_build: mm9
Supplementary_files_format_and_content: Processed files include bed files for peak predictions.
|
| |
|
| Submission date |
Jan 16, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Xiaoxiao Zhang |
| E-mail(s) |
zhangxiaoxiaozxx@gmail.com
|
| Organization name |
Harvard University
|
| Department |
Molecular and Cellular Biology
|
| Lab |
McMahon
|
| Street address |
7 Divinity Ave.
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
| |
|
| Platform ID |
GPL9250 |
| Series (2) |
| GSE43565 |
ChIP-seq analysis of β-catenin in mouse embryonic stem cells treated with GSK3 inhibitor CHIR99021 under serum+LIF standard condition. |
| GSE43597 |
Gene regulatory networks mediating canonical Wnt signal directed control of pluripotency and differentiation in embryo stem cells |
|
| Relations |
| SRA |
SRX218275 |
| BioSample |
SAMN01889183 |
