|
| Status |
Public on Aug 08, 2014 |
| Title |
H3.3 6 hrs |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic Stem Cells
|
| Organism |
Mus musculus |
| Characteristics |
time point: 6 hr
treatment: Dox
strain: 129 X C57bl/6
cell type: HA-H3.3 inducible mouseES (KH2) cells
chip antibody: HA antibody, Abcam ab9110
|
| Treatment protocol |
At the day of induction, ES cells were pre-plated on gelatin and incubated for 45 min to deplete feeder MEFs. ES cells were then split onto three gelatinized plates each of which was induced at different time points by the addition of final 2 μg/mL doxycycline hyclate (Sigma). A similar procedure was used for induction of MEFs at passage 2.
|
| Growth protocol |
ES cells were grown in standard ES medium: KO DMEM + 10% FBS, glutamine, LIF, non-essential amino acids, beta-mercaptoethanol, with mitotically inactivated feeder MEFs on gelatin coated plates until approximately 80% confluence
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Eluted ChIP materials were PCI (Phenol-Chloroform-Isoamylalcohol) extracted
ChIP material was gel purified (100 to 800bp in size) from a 2% TAE agarose gel using MiniElute columns QiaQuick (Qiagen). Gel purified DNA fragments were blunt ended and phosphorylated with an EPICENTRE End-it-Repair kit (1X buffer, 0.25mM dNTPs,1mM ATP, 1ul/50ul reaction of Enzyme mix) for 1hr at RT and cleaned up with Qiagen MiniElute spin columns. Adenosine nucleotide overhangs were added using EPICENTRE exo- Klenow for 45min at RT (with 0.2mM dATP). Illumina genome sequencing adaptors were then ligated using the EPICENTRE Fast-Link ligation kit: 11.5µl A tailed DNA eluted from a MinElute column was mixed with 1.5µl 10X ligation buffer, 0.75µl 10mMATP, 0.5µl Illumina DNA adaptors and 1µl ligase. The reaction was incubated for 1hr at RT and subsequently supplemented with 7.5 µl water, 1µl 10X buffer, 0.5µl 10mM ATP and 1µl ligase, and incubated overnight at 16οC. The ligation reaction was cleaned up with MiniElute columns (with an additional wash step to eliminate all the excess adaptors) and the adaptor ligated fragments were amplified by PCR as follows:0.75 µl of each Illumina genomic DNA sequencing primers, 6µl 10xPfx buffer 1.8µl 10mM dNTPs, 1.2µl 50mM MgSO4 and 1µl Pfx DNA polymerase (Invitrogen) were added to 30µl DNA template in a 100ul reaction. The cycling parameters were: 1. 94οC 2’; 2. 94οC 15’’; 3. 65οC 1’; 4. 68οC 30’’; 5. repeat from step 2 16 times; 6. 68οC 5’. The PCR product (250 to 450bp in size) was gel purified from a 2% TAE agarose gel using the QiaQuick columns (Qiagen). Gel purified fragments were finally precipitated with Sodium acetate and ethanol and pellets were resuspended (25nM final concentration) in TE buffer.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Chromatin IP against HA-H3.3
|
| Data processing |
Alignment by bowtie v1.0
Transform bowtie outputs into BED format by in-house scripts that extend the alignment downstream 150 or 400 bp depending on the size of the expected bound DNA fragments
Genome_build: mm9
Supplementary_files_format_and_content: bed, alignment
|
| |
|
| Submission date |
May 14, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Jui-Hung Hung |
| E-mail(s) |
juihunghung@gmail.com
|
| Organization name |
National Chiao Tung University
|
| Street address |
75 Bo-Ai Street
|
| City |
Hsin-Chu |
| ZIP/Postal code |
300 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL9250 |
| Series (1) |
| GSE57665 |
A system for genome-wide histone variant dynamics in ES cells reveals dynamic MacroH2A2 replacement at promoters |
|
| Relations |
| SRA |
SRX542619 |
| BioSample |
SAMN02777549 |
