|
| Status |
Public on Sep 02, 2017 |
| Title |
H3K9me3_T=0_Upstate |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic Stem cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: Agouti C57BL/6N substrain
cell type: Embryonic Stem cells
source: Chromatin IP against H3K9me3
antibody/details: Antibody H3K9me3 gift from Thomas Jenuwein; similar to H3K9me3 antibody Upstate/ Millipore 07-442 (lot 4861)
treatment group: T=0
|
| Treatment protocol |
Expanded heterozygous tm1a/tm2 ("T=0") ES clones were treated for 48 hours with 0.8 uM 4'hydroxytamoxifen (4'OHT, Sigma) to induce Cre activity and convert the conditional allele (tm1a) into a null allele (tm1c) ("T=2d"). These tm1c/tm2 null ES cells were subsequently cultured without 4'OHT for use in downstream assays ("T=4d"; "T=6d").
|
| Growth protocol |
JM8 mouse ES cell lines derived from the C57BL/6N strain were grown on feeder-free gelatinized tissue culture plates. The lines were maintained in Knockout DMEM (500 ml, Gibco) supplemented with 2 mM glutamine, 5 ml 100x beta-mercaptoethanol (360 ul in 500 ml PBS, filter sterilized), 10% fetal calf serum respectively (Invitrogen) and 500 U ml-1 leukaemia-inhibitory factor (ESGRO, Millipore). Trypsin solution was prepared by adding 20 ml of 2.5% trypsin solution (Gibco) and 5 ml chicken serum (Gibco) to 500 ml filter-sterilized PBS containing 0.1 g EDTA (Sigma) and 0.5 g D-glucose (Sigma).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP experiments were performed using 3.3x106 cells and 3-6 ug antibody per ChIP according to standard protocols with two minor modifications. Crosslinking of the cells was performed on the culture plates for 10 minutes, while ChIP'ed DNA was purified by Qiaquick PCR purification Kit (Qiagen). ChIP enrichment levels were analyzed by qPCR for quality control.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
ChIP DNA samples were prepared for sequencing by end repair of 20 ng DNA as measured by Qubit (Invitrogen). Adaptors were ligated to DNA fragments, which were subsequently size selected (~300bp). The adapter-modified DNA fragments were subjected to limited PCR amplification (14 cycles). Quality control was made by qPCR and by running the products on a Bioanalyzer (BioRad). Finally, cluster generation and sequencing-by-synthesis (36 bp) was performed using the Illumina Genome Analyzer IIx (GAIIx) platform according to standard protocols of the manufacturer (Illumina). The image files generated by the Genome Analyzer were processed to extract DNA sequence data. Sequences were aligned to the mouse MM9 reference genome using the Illumina Analysis Pipeline allowing one mismatch. Samples were sequenced to a depth of approximately 10-20 million mapped tags per sample. Unless specified otherwise, only the tags uniquely aligning to the genome were considered for further analysis. The 36 bp sequence reads were directionally extended to 300 bp, corresponding to the length of the original fragments used for sequencing, and tags mapping on exact the same genomic locus were discarded to obtain a non-redundant set. The output data were converted to Browser Extensible Data (BED) files for downstream analysis and Wiggle (WIG) files for viewing the data in the UCSC Genome Browser. All ChIP-Seqsequence analyses were conducted based on the Mus musculus NCBI m37 genome assembly (MM9) accessed from the Ensembl (release 54; May 2009) or the UCSC (assembly July 2007) Genome Browsers.
|
| |
|
| Submission date |
Aug 31, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Hendrik Marks |
| E-mail(s) |
[email protected]
|
| Organization name |
Radboud University Nijmegen, RIMLS
|
| Department |
Molecular Biology
|
| Street address |
Geert Grooteplein 26/28
|
| City |
Nijmegen |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE31777 |
An efficient method for generation of bi-allelic null mutant mouse embryonic stem cell lines and its application for investigating epigenetic modifiers |
|
| Relations |
| SRA |
SRX095326 |
| BioSample |
SAMN00715020 |
