|
| Status |
Public on Nov 04, 2009 |
| Title |
H3K9me3-Eset shRNA_ChIPseq |
| Sample type |
SRA |
| |
|
| Source name |
embryonic stem cells, Eset shRNA
|
| Organism |
Mus musculus |
| Characteristics |
cell line: E14
cell type: embryonic stem cells
knockdown: Eset
antibody: anti-H3K9Me3 antibody
|
| Treatment protocol |
The ES cells were fixed in 1% formaldehyde for 10 minutes at room temperature. The formaldehyde was quenched using 0.1 M glycine before harvest for extract preparation.
|
| Growth protocol |
E14 mouse ES cells, cultured under feeder-free conditions, were maintained in Dulbecco's Modified Eagle-Medium (DMEM, GIBCO), with 15 % heat-inactivated ES qualified fetal bovine serum (FBS, GIBCO), 0.055 mM beta-mercaptoethanol (GIBCO), 2mM L-glutamine, 0.1 mM MEM non-essential amino acid, 5,000 units/ml penicillin/streptomycin and 1,000 units/ml of LIF (Chemicon). Control shRNA and Eset shRNA knockdown samples were obtained by transfecting ES cells with the respective shRNAs using lipofectamine-2000(invitrogen). The transfected ES cells were maintained in the ES medium with 0.8ug/ml puromycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Whole cell extracts were sonicated to solubilize the chromatin. The chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using different antibodies. ChIP-enriched DNA from multiple ChIP experiments was pooled and quantified using PicoGreen dsDNA quantitation kit (Invitrogen). The ChIP-enriched DNA was processed for Solexa sequencing using ChIP-Seq Sample Prep Kit (Illumina).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
Anti-H3K9Me3 (ab8898; Abcam) antibody was used for ChIP of Eset shRNA knockdown sample.
A pool of 3 ChIP samples was used.
|
| Data processing |
The raw images have been processed using the Solexa Pipeline and mapped to the reference genome (NCBI Build 36, mm8) using Eland software with maximal 2 mis-matches.
The control knockdown and Eset knockdown cells were directly compared for H3K9me3 enrichment, and the regions that were enriched in the control sample were identified.
The following supplementary files contain alignments:
GSM440258_CME037_R00087_lane1_s_1_sorted.txt
GSM440258_CME037_R00087_lane2_s_2_sorted.txt
|
| |
|
| Submission date |
Aug 13, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Mikael Huss |
| E-mail(s) |
hussem@gis.a-star.edu.sg
|
| Phone |
+6564788042
|
| Organization name |
Genome Institute of Singapore
|
| Street address |
60 Biopolis Street
|
| City |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL9185 |
| Series (1) |
| GSE17642 |
Genome-wide mapping of Eset-binding sites and H3K9me3 state in mouse embryonic stem cells |
|
| Relations |
| SRA |
SRX014435 |
| BioSample |
SAMN00006250 |
