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Sample GSM4145820 Query DataSets for GSM4145820
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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