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| Status |
Public on Apr 25, 2022 |
| Title |
Bap1 cKO H3K27-me3 rep1 |
| Sample type |
SRA |
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| Source name |
endosteal mesenchymal stromal cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
genotype: Bap1 cKO (Prx1-cre;Bap1fl/fl)
antibody: anti-trimethyl-H3K27 (Upstate)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To cross-link DNA to histones, 10^6 endosteal MSCs were harvested and incubated with a 1% formaldehyde solution for 10 min at 37°C on a rocker. After fixation, the cells were lysed with EzRIPA lysis buffer (150mM NaCl, 0.1% SDS, 1% NP-40, 0.5% Deoxycholic acid, 20mM HEPES pH7.5, ATTO) supplemented with a protease inhibitor and sonicated using a Bioruptor to make an average of 300~500 bp length. For pre-clearing, the sonicated cell supernatants were diluted with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH8.1, 167 mM NaCl) and incubated with Protein A Agarose/Salmon Sperm DNA (Millipore) for 30 min at 4°C on a rocker. Then, the samples were incubated with anti-ubiquityl-H2AK119 (Cell signaling), anti-trimethyl-H3K27 (Upstate), anti-acetyl-H3K27 (Upstate), and anti-trimethyl-H3K4 (Abcam) antibodies overnight at 4°C on a rocker. The beads were consecutively washed twice with a low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), a high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), LiCl washing buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1), and TE buffer (10 mM Tris pH 8, 1 mM EDTA). After washing of the beads, DNA-histone complexes were eluted with elution buffer (1% SDS, 0.1M NaHCO3) and were incubated with 5M NaCl for 4h at 65°C to reverse DNA-histone cross-linking. Then, DNA was purified using QIAquick Spin Kit (Qiagen). Purified DNA was analyzed by real-time quantitative PCR using Step One Plus (Applied Biosystems).
The ChIP-seq libraries were prepared using TruSeq DNA Sample prep Kit (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The adapter sequences were trimmed using cutadapt v.1.18. Remaining reads were then aligned to the mouse genome (GRCm38) using Bowtie2 v.2.4.5 with the default parameters. PCR or optical duplicate reads, multi-mapped reads, and the reads aligned with mapping quality (MAPQ) < 5 were filtered out using Picard v.2.25.5 and Samtools v.1.9.
The genomic region between TSS-5kb and TSS+5kb was binned into 50-bp bins, and the number of reads in each bin was counted using the coverage tool from bedtools v.2.30.0 with the default parameters.
The read counts were then converted to counts per million (CPM) based on the library size.
Assembly: GRCm38
Supplementary files format and content: tab-delimited text files including read counts in individual bins parsed from genomic regions of differentially expressed genes for each sample
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| Submission date |
Mar 15, 2022 |
| Last update date |
Apr 25, 2022 |
| Contact name |
Do Young Hyeon |
| E-mail(s) |
doyoungh@postech.ac.kr
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| Organization name |
Seoul National University
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| Department |
School of Biological Sciences
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| Lab |
Systems Biology and Medicine Laboratory
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| Street address |
1 Gwanak-ro, Gwanak-gu
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| City |
Seoul |
| State/province |
Select State |
| ZIP/Postal code |
08826 |
| Country |
South Korea |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE198649 |
Bap1 shapes the bone marrow niche for lymphopoiesis by fine-tuning epigenetic profiles in endosteal mesenchymal stromal cells [ChIP-Seq] |
| GSE198824 |
Bap1 shapes the bone marrow niche for lymphopoiesis by fine-tuning epigenetic profiles in endosteal mesenchymal stromal cells |
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| Relations |
| BioSample |
SAMN26669439 |
| SRA |
SRX14466878 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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