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| Status |
Public on May 21, 2024 |
| Title |
ChIP_Sp5_gastruloids_1 |
| Sample type |
SRA |
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| Source name |
E14_triple reporter
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| Organism |
Mus musculus |
| Characteristics |
chip antibody: HA
cell line: E14_triple reporter
cell type: gastruloids
genotype: endogenous Sp5-HA-FKBP12F36V degron tag
assay: ChIP-seq
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| Growth protocol |
The dual BRA-GFP (Brachyury), SOX17-RFP reporter mouse ES cell line (Pour et al., 2022 ) was cultured on 0.15% (w/v) gelatin-coated dishes in Dulbeccco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 15% fetal bovine serum (FBS, HyClone), 2mM GlutaMAX (Gibco), non-essential amino acids (Gibco), 50U/mL penicillin-streptomycin (Gibco), 100µM β-mercaptoethanol (Sigma) and leukemia inhibitory factor (LIF; produced in-house). Gastruloids were generated as previously described (Protoc. Exch. https://doi.org/10.1038/protex.2018.094, van den Brink et al., Nature 2020). Briefly, 300 single cells were FACS sorted with a BD FACSMelody™ cell sorter into U-bottomed 96 wells (Greiner Bio-one; 650185) containing 40μL NDiff227 (Takara). After 48h, the aggregates were treated with 3μM CHIR99021 (Axon 1386) and medium was refreshed every day with pre-equilibrated NDiff227.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Ttrypsinized gastruloids were crosslinked in solution A with 2mM disuccinimidyl glutarate (Thermo Fisher Scientific #20593) for 25 min, followed by 1% formaldehyde methanol-free (Thermo Fisher Scientific, #28906) for 20 min. Chromatin extracts were sonicated for 2-4 cycles of 30 sec on, 30 sec off using a Diagenode Bioruptor Pico. For each ChIP, 4µg of V5 Tag antibody (Thermo Fisher Scientific, #R960CUS) or 4µg of HA antibody (a mix of 2µg from Roche 11867423001 and 2µg from Invitrogen 71–5500) was pre-conjugated to 40ul of Protein A/G magnetic beads (Invitrogen).
Immunoprecipitated DNA was processed for library preparation using the Kapa HyperPrep Kit (Kapa Biosystems) and barcoded with NEXTflex DNA barcode.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Sequence reads were processed using the seq2science pipeline (v0.7.1, https://github.com/vanheeringen-lab/seq2science). Briefly, paired-end reads were trimmed with fastp (v0.20.1, default settings) and aligned with bwa-mem2 (v2.2.1, options ‘-M’) to the Mus musculus genome assembly GRCm38.p6. Aligned reads were filtered based on mapping quality (MAPQ ≥ 30) and filtering out duplicate reads and reads in the ENCODE blacklisted regions. Peaks were called with MACS2 (v2.2.7) with default settings in BAMPE mode. The peaks called in both replicates were used for analysis.
Assembly: mm10
Supplementary files format and content: narrowPeak
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| Submission date |
Apr 05, 2023 |
| Last update date |
May 21, 2024 |
| Contact name |
Suzan Stelloo |
| E-mail(s) |
Stelloo@science.ru.nl
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| Organization name |
Radboud University
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| Department |
Molecular Biology
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| Street address |
Geert Grooteplein Zuid 28
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| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE229024 |
Deciphering lineage specification during early embryogenesis in mouse gastruloids using integrative multi-layered proteomics [ChIP-seq] |
| GSE229029 |
Deciphering lineage specification during early embryogenesis in mouse gastruloids using integrative multi-layered proteomics |
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| Relations |
| BioSample |
SAMN34081637 |
| SRA |
SRX19884807 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7147974_GRCm38.p6-095_Suz_ChIP_SP5_1_Z44_S23_peaks.narrowPeak.chr.narrowPeak.gz |
533.8 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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