|
| Status |
Public on Apr 29, 2024 |
| Title |
Sample_H3K27me3_shDaxx |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T_shRNA-Daxx_H3K27me3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK 293T
cell type: human embryonic kidney cells
transfected with: shRNA specific to human Daxx
passages: 10-15
chip antibody: H3K27me3
chip antibody vendor: Millipore
chip antibody cat. #: 07-449
chip antibody lot #: JBC1873477
|
| Treatment protocol |
In order to knock down Daxx, HEK293T subjected to be transfected with lentivirus plasmid constructed with sort hairpin RNA target to Daxx and luciferase, respectively.
|
| Growth protocol |
HEK293T cells were cultured in DMEM (high glucose, Gibco) supplemented with 10 % fetal bovine serum (Gibco), 10 mM NaHCO3 (Sigma) and penicillin/streptomycin (Gibco).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell lysis extracts were sonicated and immunoprecipitated with 5ug H3K27me3 antibody (Merk 07-449). DNA was purified and measured by Nano drop 2000 spectrophotometer (Thermo). Total 30ng of DNA was used for preparation the ChIP-Seq DNA library following by ChIP-Seq Sample Prep Kit (Illumina). Briefly, the kit converted the DNA overhangs into phosphorylated blunt ends, using T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase), and T4 polynucleotide kinase (PNK). The 3' to 5' exonuclease activity of these enzymes removed 3' overhangs and the polymerase activity filled in the 5' overhangs. Subsequently, the protocol added an ‘A’ base to the 3' end of the blunt phosphorylated DNA fragments, using the polymerase activity of Klenow fragment (3' to 5' exo minus). This prepared the DNA fragments for ligation to the adapters, which have a single ‘T’ base overhang at their 3' end. After adaptor ligation, adequate DNA were selected from gel and subjected to PCR amplification. High throughput sequencing was performed by the Hiseq 2000 (Illumina) as manufacturer’s protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
the processd data obtained from this file is compared with the input(Input_D)
|
| Data processing |
Basecalls performed using CASAVA version 1.8
ChIP-seq reads were aligned to the hg19 genome assembly using bwa version 0.6.1 with the following confiuration "bwa aln -q 20"
Data were filtered if mapping quality is lower than 20.
peaks were called using SICER version 1.1 with the following setting: window size 200, gap size 600, FDR 0.01
The islands in two pairs of liraries were identified by SICER-df.sh and were compared with each other to determine the significant change.
Genome_build: hg19
Supplementary_files_format_and_content: wig files were generated using SICER
|
| |
|
| Submission date |
May 23, 2012 |
| Last update date |
Apr 29, 2024 |
| Contact name |
Hsiu-Ming Shih |
| E-mail(s) |
hmshih@ibms.sinica.edu.tw
|
| Organization name |
Academia Sinica
|
| Department |
Institute of Biomedical Sciences
|
| Street address |
128 Sec. 2, Academia Rd. Nankang,
|
| City |
Taipei |
| ZIP/Postal code |
11529 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE38184 |
Genome-wide mapping of H3K27me3 in Daxx-depleted cells |
|
| Relations |
| SRA |
SRX149228 |
| BioSample |
SAMN00997891 |
