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Sample GSM4954699 Query DataSets for GSM4954699
Status Public on Dec 05, 2020
Title young HSC input rep2
Sample type SRA
 
Source name 10 week hematopoietic stem cells
Organism Mus musculus
Characteristics cell type: Murine bone marrow derived HSCs
surface markers: IL-7- Lin- Sca-1+ c-kit+ CD34- CD150+ CD48- Flt3-
age: 10-week-old
chip antibody: none
strain: C57BL/6
Extracted molecule genomic DNA
Extraction protocol Cells were cross-linked with 0.5% formaldehyde at 37°C for 2 min, washed with PBS containing 2% FBS, lysed with ChIP buffer (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM CaCl2, 0.5% NP40, and protease inhibitor cocktail)
Extracted samples were sonicated for 5 sec × 3 times (90 sec on ice) using a Bioruptor (Cosmo Bio). Cells were digested with micrococcal nuclease (MNase) (NEB) at 37°C for 40 min and treated with 10 mM EDTA to stop the reaction. After the addition of an equal volume of RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS), cells were resonicated for 5 sec × 10 times (300 sec on ice) using the Bioruptor. After centrifugation, the soluble chromatin fraction was immunoprecipitated at 4°C overnight with Dynabeads M-280 Sheep anti-Rabbit IgG conjugated with an anti-H3K27me3 (07-449, Millipore), anti-H2AK119ub1 (8240, Cell Signaling Technology),or anti-H3K4me3 (07-473, Millipore) antibody. Immunoprecipitates were washed with high salt ChIP buffer (500 mM NaCl) four times and TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA) twice. Bound chromatin and 25 μl of input DNA were suspended in 47.5 and 22.5 μl of elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, and 250 mM NaCl), respectively, and vortexed. After the addition of 5 μl of 5 M NaCl, the solutions were incubated at 65°C for 4 hr and then treated with 25 μg/ml RNase A (Sigma-65 Aldrich) at 37°C for 30 min and 0.1 mg/ml proteinase K (Roche) at 50°C for 1 hr to reverse formaldehyde crosslinking. Eluted DNA was purified with a MinElutePCR Purification Kit (Qiagen).DNA libraries were prepared from input and ChIP DNA samples using a ThruPLEX DNA-seq Kit (Rubicon Genomics) according to the manufactures’instructions. DNA libraries were quantified using high sensitivity Chip on the Bioanalyzer (Agilent) and sequenced with Hiseq1500 (Illumina) according to the manufacturer’s instructions. Sixty-one cycles of sequencing reactions were performed.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 1500
 
Data processing alignment:sequences were aligned to mouse genome sequences (mm10) with Bowtie2(default setting). Only uniquely mapped reads were retained for downstream analysis
We used macs2(ver2.2.6) to detect peak of histone modification with nomodel option
For H3K4me3, narrow peaks with a q-value cutoff of 0.05 was adopted. For H3K27me3, broad peaks with a q-value cutoff of 0.10 was adopted.
For visualization to make bigwig files,reads per million mapped read (RPM) values of the sequenced reads were calculated every 2,000-base pair bin with a shifting size of 200 base pairs using bed tools and normalized with the amount of ChIP DNA in the H3K27me3 analysis.
Genome_build: mm10
 
Submission date Dec 02, 2020
Last update date Dec 05, 2020
Contact name Atsushi Iwama
Organization name The Institute of Medical Science, The University of Tokyo
Department Center for Stem Cell Biology and Regenerative Medicine
Lab Division of Stem Cell and Molecular Medicine
Street address 4-6-1, Shirokanedai
City Minato-ku
State/province Tokyo
ZIP/Postal code 108-8639
Country Japan
 
Platform ID GPL18480
Series (2)
GSE162570 Epigenetic traits inscribed in chromatin accessibility in aged hematopoietic stem cells (ChIP-seq H3K27me3 H3K4me3)
GSE162662 Epigenetic traits inscribed in chromatin accessibility in aged hematopoietic stem cells
Relations
BioSample SAMN16984651
SRA SRX9621930

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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