 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 06, 2020 |
| Title |
GS_control_IgG_crosslink3 |
| Sample type |
SRA |
| |
|
| Source name |
GS_control
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Germline stem (GS) cell line
Sex: male
treatment: control
antibody: normal rabbit IgG (Upstate 12-370)
|
| Growth protocol |
Germline stem (GS) cells (kindly provided by Dr Takashi Shinohara) were cultured on mitomycin C treated primary embryonic fibroblast (PEF) cells according to the previous report (Kanatsu-Shinohara et al., Biol Reprod., 2003, 69, 612-616) with minor modifications. Before GS cell sampling, PEF cells were removed by a differential attachment method to gelatin coated dishes following standard procedures
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For crosslink1, cells were fixed in 2 mM disuccinimidyl glutarate, 1.5 mM ethylene glycolbis succinimidyl succinate in PBS followed by 1% formaldehyde. Crosslinked cells were lysed in 1% SDS, 10 mM EDTA, 50 mM Tris with protease inhibitors by sonication with Covaris S220. ChIP was performed by using normal rabbit IgG (Upstate 12-370) and protein A/G agarose beads. For crosslink2, cells were fixed in 0.5% formaldehyde in PBS. Crosslinked cells were first lysed in 0.3M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 15 mM Tris, 0.5 mM DTT, 0.5% TritonX-100 with protease inhibitors, then nuclei were pelleted by centrifugation of the above lysates on 1.2 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 15 mM Tris, 0.5 mM DTT. Nucleosome fractions were obtained by MNase treatment. ChIP was performed by using normal rabbit IgG (Upstate 12-370) and protein A/G agarose beads. For crosslink3, cells were fixed in 1% formaldehyde in PBS. Crosslinked cells were lysed in 1% SDS, 10 mM EDTA, 50 mM Tris with protease inhibitors by sonication with Covaris S220. ChIP was performed by using normal rabbit IgG (Upstate 12-370) and protein A/G agarose beads. Eluates were treated with RNase A and Proteinase K followed by phenol/chloroform extraction and ethanol precipitation of DNA.
Libraries were constructed from control IgG ChIP DNA or genomic DNA (from whole cell extract) using TruSeq ChIP Sample Preparation Kit (Illumina) and sequenced by MiSeq with MiSeq Reagent Kit v3 (Illumina).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
control IgG ChIP
|
| Data processing |
ChIPseq reads were aligned to the mouse genome (mm10) using Bowtie2 2.3.4.1.
Macs2 2.1.0 callpeak and bedGraphcomp were used to process aligned ChIPseq reads.
Genome_build: mm10
Supplementary_files_format_and_content: bedGraph files were generated by Macs2 2.1.0. Data represent fold enrichment scores relative to control lambda values.
|
| |
|
| Submission date |
Oct 31, 2019 |
| Last update date |
Feb 07, 2020 |
| Contact name |
Shinichiro Chuma |
| E-mail(s) |
chuma.shinichiro.3x@kyoto-u.ac.jp
|
| Phone |
+81-75-751-3821
|
| Organization name |
Kyoto university
|
| Department |
Institute for Frontier Life and Medical Sciences
|
| Lab |
Laboratory of Developmental Epigenome
|
| Street address |
53 Kawahara-cho, Shogoin, Sakyo-ku
|
| City |
Kyoto |
| State/province |
Kyoto |
| ZIP/Postal code |
606-8507 |
| Country |
Japan |
| |
|
| Platform ID |
GPL16417 |
| Series (2) |
| GSE116798 |
Transcriptomic and epigenetic profiling of genome control in the germline stem cell cycle in mice |
| GSE139677 |
Transcriptomic and epigenetic profiling of genome control in the germline stem cell cycle in mice [ChIP-seq II] |
|
| Relations |
| BioSample |
SAMN13170242 |
| SRA |
SRX7081140 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4145820_GS_control_IgG_crosslink3_FE.bedGraph.gz |
87.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
