 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 21, 2011 |
| Title |
TT2_WT_H3K9me3_NChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
Embyonic Stem Cell
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Embyonic Stem Cell
strain: TT2 (C57BL/6/CBA F1)
genotype/variation: WT
chip antibody: H3K9me3 (Active Motif, cat #39161, lot #170)
|
| Treatment protocol |
none
To generate chromatin, 1 x 107 infected ES cells for each cell line were harvested and washed in PBS. Cells were resuspended in 250 ul of douncing buffer (10 mM Tris-HCl, pH 7.5, 4 mM MgCl2, 1 mM CaCl2, 1X Protease inhibitory cocktail (PIC) and homogenized through a 25 G5/8 needle syringe for 20 repetitions. 1.875 ul of 20 U/ul of MNase (Worthington Biochemicals) was added and incubated at 37 0C for 7 min. The reaction was quenched by 0.5 M EDTA and incubated on ice for 5 min. 1ml of hypotonic buffer (0.2 mM EDTA, pH 8.0, 0.1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 1.5 mM dithiothreitol, 1X PIC) was added and incubated for 1 hour on ice. Cellular debris was pelleted and the supernatant was recovered.
|
| Growth protocol |
J1 and TT2 ES cells were passaged every 48-72 hours in DMEM supplemented with 15% FBS (HyClone), 20mM HEPES, 0.1mM nonessential amino acids, 0.1mM 2- mercaptoethanol, 100 units/ml penicillin, 0.05 mM streptomycin, leukemia inhibitory factor (LIF) and 2mM glutamine on gelatinized plates.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was divided into aliquots of 2 x 106 cell equivalents per IP tube and the volume adjusted to 325 ul with IP buffer. Three aliquots were used for H3K9me3 immunoprecipitation and 1 aliquot was used for control IgG immunoprecipitation. Antibodies specific for H3K9me3 (5 ul; Active Motif, 39161), H3K27 (07-449, Millipore) or control IgG (20 ug; Sigma I8140) were added to each tube and rotated at 4 oC for 1 hour. The antibody-protein-DNA complex was precipitated by adding 20 ul of the blocked protein A/G beads and rotated at 4 0C overnight. The immunoprecipitated complex was washed twice with 400 ul of ChIP Wash buffer (20 mM Tris-HCl, pH 8.0, 0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 150 mM NaCl, 1X PIC), followed by a single wash with ChIP Final Wash Buffer (20 mM Tris-HCl (pH 8.0), 0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 500 mM NaCl, 1X PIC). The protein-DNA complex was eluted by incubating the beads in 200 ul of elution buffer (100 mM NaHCO3, 1% SDS) for 2 hours at 680C. Immunoprecipitated material was purified using the QIAquick PCR Purification Kit (Qiagen), according to the manufacturers protocol.
Sheared cDNA or IPed material was resolved on a 12% acrylamide gel and the 100-300bp size fraction was eluted from the gel slice overnight at 4oC in 300 ml of elution buffer (5:1, LoTE, 7.5M Ammonium Acetate). DNA was recovered from the resulting gel slurry using a QIAquick PCR Purification Kit (Qiagen). Following column elution, the DNA ends were repaired using a 1:5 mixture of T4 and Klenow DNA polymerases (New England Biolabs), purified by phenol chloroform isoamyl alcohol (pH 8.0, 100ul, Fisher) extraction and EtOH precipitated. A single "A" base was added to the DNA fragments using Klenow exo- (3' and 5' exo minus) (New England Biolabs). Adapters (Illumina) were ligated to ends of the "A"-tailed DNA and adapter-modified DNA fragments enriched by PCR using Phusion polymerase (Finnzymes) and PCR primer 1.1 and 2.1 (Illumina) following the manufacturer's instructions (15 PCR cycles). The resulting PCR product was purified using a 8% PAGE (Nuvex; Invitrogen, Burlington, ON, Canada) to remove small products including adapter dimers, and the DNA quality assessed using an Agilent DNA 1000 series II assay and quantified by Qubit fluorometer (Invitrogen, Burlington, ON, Canada) and then diluted to 10nM. Cluster generation and 51 or 76 cycles of sequencing were performed on the Illumina cluster station and Illumina Genome AnalyzerIIx sequencing platform
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Chromatin IP against H3K9me3
|
| Data processing |
Alignment (ChIP-seq): Sequence reads were mapped to mm9 (NCBI 37) using MAQ (v 0.7.1) (Li et al., 2008) and default parameters.
Wigs (ChIP-seq):Reads having identical coordinates were collapsed into a single read and reads with mapQ >=7 passed to FindPeaks 3.1 (Fejes et al., 2008) (with a fixed directional read extension of 150bp) for segmentation and visualization
|
| |
|
| Submission date |
May 20, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Eric Chuah |
| E-mail(s) |
echuah@bcgsc.ca
|
| Phone |
604-707-5900 3231
|
| Organization name |
BC Genome Sciences Centre
|
| Street address |
570 West 7th Ave
|
| City |
Vancouver |
| State/province |
BC |
| ZIP/Postal code |
V5Z 4S6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL9250 |
| Series (1) |
| GSE29413 |
DNA methylation and SETDB1/H3K9me3 regulate predominantly distinct sets of genes, retroelements and chimaeric transcripts in mouse ES cells |
|
| Relations |
| SRA |
SRX063938 |
| BioSample |
SAMN00619176 |
| Named Annotation |
GSM727425.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM727425.bam |
1.5 Gb |
(ftp)(http) |
BAM |
| GSM727425.wig.gz |
47.1 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
