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| Status |
Public on Dec 05, 2020 |
| Title |
young HSC input rep1 |
| Sample type |
SRA |
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| Source name |
10 week hematopoietic stem cells
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| 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
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| 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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
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| 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
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| 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
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| Department |
Center for Stem Cell Biology and Regenerative Medicine
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| Lab |
Division of Stem Cell and Molecular Medicine
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| Street address |
4-6-1, Shirokanedai
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| City |
Minato-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
108-8639 |
| Country |
Japan |
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| 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 |
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| Relations |
| BioSample |
SAMN16984654 |
| SRA |
SRX9621928 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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