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| Status |
Public on Jul 28, 2020 |
| Title |
H2AK119ub_HPC7 |
| Sample type |
SRA |
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| Source name |
HPC7 cell line stably expressing triple-FLAG-tagged murine MYSM1
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| Organism |
Mus musculus |
| Characteristics |
cell type: HPC7 cell line
chip antibody: anti-H2AK119Ub (Cell Signaling Technology, D27C4, Rabbit monoclonal)
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| Growth protocol |
The Ba/F3 cell line stably expressing triple-Flag-tagged MYSM1 was maintained at 0.5-2 x 10^6 cells/mL in RPMI-1640 (Wisent) with 10% Fetal Calf Serum (FCS, Wisent), 2mM L-Glutamine, 100μg/mL streptomycin, 100U/mL penicillin (Wisent), 5% WEHI conditioned media as the source of IL-3, and under 2μg/mL puromycin selection (Wisent). The HPC7 cell line stably expressing triple-Flag-tagged MYSM1 was cultured at 0.5-2 x 10^6 cells/mL in IMDM (Life Technologies), with 10% FCS (Gibco, Life Technologies), 100μg/mL streptomycin, 100U/mL penicillin (Wisent), 7.48x10^-5M MTG (M6145, Sigma-Aldrich), 100 ng/mL murine SCF (Shenandoah Biotechnology, Cedarlane), and under 2μg/mL puromycin selection (Wisent).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked for 10 min at room temperature with 1% formaldehyde added in culture medium. Crosslink was stopped with ice-cold PBS containing 0.125M glycine for 5min. Nuclei were prepared by sequential incubation on ice for 5min in buffer A [10mM Tris-HCl (pH 8), 10mM EDTA, 0.25% Triton X-100], and for 30min in buffer B [10mM Tris-HCl (pH 8), 1mM EDTA, 200mM NaCl] (all buffers include proteases inhibitors). Nuclei were resuspended in sonication buffer [10mM Tris-HCl (pH 8), 1mM EDTA, 0.5% SDS, 0.5% Triton X-100, 0.05% NaDOC, 140mM NaCl] and sonicated with a Branson Digital Sonifier (Branson Ultrasonics) to an average size of 250bp (12 cycles of 30 seconds at 80%, with 30 seconds rest in cooled circulating water). Sonicated chromatin from the equivalent of 5x10^6 cells per ChIP was incubated overnight on a rotating platform at 4°C with 40μL of Dynabeads Protein G (Invitrogen, Life Technologies) conjugated to 3-5μg of antibodies: anti-Flag M2 (Sigma, F1804, for MYSM1-FLAG ChIP), anti-H3K27Ac (Abcam, ab4729) or anti-H2AK119Ub (Cell Signaling Technology, D27C4, Table S10). Immune complexes with anti-Flag M2 antibody was washed sequentially for 2min at room temperature with 1ml of the following buffers: Wash 1 [0.5% NP-40, 150mM KCl, 1mM EDTA, 10mM Tris-HCl pH8]; Wash 2 [0.5% Triton, 0.1% NaDOC, 100mM NaCl, 10mM Tris-HCl pH8]; Wash 3A [0.5% Triton, 0.1% NaDOC, 400mM NaCl, 10mM Tris-HCl pH8]; Wash 3B [0.5% Triton, 0.1% NaDOC, 500mM NaCl, 10mM Tris-HCl pH8]; and Wash 4 [0.5% NaDOC, 0.5% NP-40, 250mM LiCl, 1mM EDTA, 10mM Tris-HCl pH8]. Immune complexes with anti-H3K27Ac and anti-H2AK119Ub antibodies were washed sequentially for 2min at room temperature with 1ml of the following buffers: Wash B [1% Triton X-100, 0.1% SDS, 150mM NaCl, 2mM EDTA, 20mM Tris-HCl pH 8]; Wash C [1% Triton X-100, 0.1% SDS, 500mM NaCl, 2mM EDTA, 20mM Tris-HCl pH 8]; Wash D [1% NP-40, 250mM LiCl, 1mM EDTA, 10mM Tris-HCl pH 8]; and TEN buffer [50mM NaCl, 10mM Tris-HCl pH 8, 1mM EDTA]. After de-crosslinking by overnight incubation at 65°C, the ChIP and Input DNA was purified with Qiaquick PCR Cleanup kit following manufacturer’s instructions (Qiagen).
ChIP-seq libraries were prepared using the Illumina TruSeq kit and sequenced on an Illumina HiSeq 2500 sequencer using a 50bp paired-end configuration. Sonicated input DNA from the same cells were sequenced as negative control.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Reads quality filtered according to the Illumina pipeline
Experimental and input control sequence reads were mapped to the mouse mm9 reference genome assembly with Bowtie 1.0.0 using the following settings: --best --trim5 2 mm9.
Chromatin binding sites of MYSM1 were identified using peak detection algorithm MACS (version 1.4.1, default parameters), with comparisons for read enrichment against control input DNA from the same cells.
The resulting binding sites were filtered to remove regions with < 5 fold enrichment compared to the input DNA and < 5 normalized tag counts within 200bp to the peak summit.
Genome_build: mm9
Supplementary_files_format_and_content: filtered bedfile of MYSM1 peak summits
Supplementary_files_format_and_content: bigWig
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| Submission date |
May 15, 2020 |
| Last update date |
Jul 29, 2020 |
| Contact name |
David Langlais |
| E-mail(s) |
[email protected]
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| Organization name |
McGill University
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| Department |
Human Genetics
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| Lab |
Inflammation Genomics Lab
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| Street address |
740 Ave Dr Penfield, rm 4203, McGill Genome Centre
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| City |
MONTREAL |
| State/province |
QC |
| ZIP/Postal code |
H3A0G1 |
| Country |
Canada |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE150663 |
MYSM1 maintains ribosomal protein gene expression in hematopoietic stem cells to prevent hematopoietic dysfunction I. |
| GSE150667 |
MYSM1 |
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| Relations |
| BioSample |
SAMN14933595 |
| SRA |
SRX8349076 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4555941_H2AK119ub_HPC7_norm1e7.bw |
646.8 Mb |
(ftp)(http) |
BW |
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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