|
| Status |
Public on Apr 21, 2011 |
| Title |
mES_5hmC_Luc_shRNA |
| Sample type |
SRA |
| |
|
| Source name |
ES cells, Luc shRNA, hMeDIP
|
| Organism |
Mus musculus |
| Characteristics |
cell line: E14
cell type: mouse embryonic stem (MES) cells
shrna treatment: Luc shRNA
antibody: rabbit anti-5hmC antibody
antibody vendor: Active Motif
antibody catalog number: 39769
antibody lot number: 10310001
|
| Treatment protocol |
E14 ES cells were infected with Luc shRNA containing lentivirus and harvested after 4-days selection with puromycin.Genomic DNA was purified and sonicated. Illumina adapters were ligated before hMeDIP. 5µg of adapter-ligated DNA was denatured and incubated with 5µl of 5hmC antibody (Active Motif) at 4oC overnight. Antibody-DNA complexes were captured by protein A/G beads. The immunoprecipitated DNA was purified and sequenced followed by standard Illumina protocols.
|
| Growth protocol |
E14 ES cells were feeder-free cultured.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was end repaired using the End-It DNA End-repair kit (Epicentre), and purified with the QiaQuick PCR purification kit. DNA was treated with Klenow exo- (NEB) in NEB buffer 2 with 1mM ATP. After incubating at 37oC for 50min, the "A" base added DNA was purified with the QiaQuick PCR purification kit. Illumina adapters were ligated using quick T4 DNA ligase. The final DNA was purified with the QiaQuick PCR purification kit and amplified using Illumina sequencing primers for 16-18 cycles.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
hMeDIP-seq
hMeDIPed genomic DNA.
|
| Data processing |
We used Off-Line Basecaller software (Illumina v1.8) to analyze images and call bases, and used the CASAVA package (Illumina v1.6) to generate read sequences and align to mm9. Since the DNA fragment size in our library is mainly from 200-300bp, we then extended the sequence to 250bp downstream of the 5' end of reads and converted the sorted file into bed format.
|
| |
|
| Submission date |
Apr 11, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Feizhen Wu |
| E-mail(s) |
wufz@fudan.edu.cn
|
| Phone |
86-21-54237821
|
| Organization name |
Fudan Univ, Shanghai, China
|
| Department |
Institutes of Biomedical Sciences
|
| Lab |
Epigenetics lab
|
| Street address |
Dongan Road 131, Rm 511 Mingdao Building
|
| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
200032 |
| Country |
China |
| |
|
| Platform ID |
GPL9185 |
| Series (2) |
| GSE28500 |
Genome-wide Regulation of 5hmC, 5mC and Gene Expression by Tet1 Hydroxylase in Mouse Embryonic Stem Cells |
| GSE28532 |
Genome-wide Regulation of 5hmC, 5mC and Gene Expression by Tet1 Hydroxylase in Mouse Embryonic Stem Cells (ChIP-seq data) |
|
| Relations |
| SRA |
SRX057747 |
| BioSample |
SAMN00259669 |
| Named Annotation |
GSM706677_mES_5hmC_Luc_shRNA.bed.gz |
