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| Status |
Public on Aug 21, 2024 |
| Title |
WT_E14, Embryonic stem cells, RAD21 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Embryonic stem cells
genotype: Wild_type
cell line: E14
chip antibody: RAD21 (Abcam, ab217678)
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| Growth protocol |
mESCs were cultured in standard medium supplemented with LIF and 2i conditions (1 mM MEK1/2 inhibitor (PD0325901, Stemgent) and 3 mM GSK3 inhibitor (CHIR99021, Stemgent)). For motor neuron (MN) differentiation, the previously described protocol in Narendra et. al. (2015) was applied. Briefly, ESCs were differentiated into embryoid bodies in 2 days (Day 0) in ANDFK medium (Advanced DMEM/F12 : Neurobasal (1:1) Medium, 15% Knockout Serum Replacement, Pen/Strep, 2 mM L-Glutamine, and 0.1 mM 2-mercaptoethanol). Medium was exchanged on Day 2 of differentiation, and further patterning of embryoid bodies was induced by supplementing the ANDFK media on Day 2 with 1 μM all-trans-Retinoic acid (RA, Sigma) and 0.5 μM smoothened agonist (SAG, Calbiochem).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed as described in Oksuz et al., 2018 using buffers in the following order: LB1 (50 mM HEPES, pH 7.5 at 4 C, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% Triton X) for 10 min at 4 C, LB2 (10 mM Tris, pH 8 at 4 C, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) for 10 min at RT, and LB3 (10 mM Tris, pH 7.5 at 4 C, 1 mM EDTA, 0.5 mM EGTA, and 0.5% N-Lauroylsarcosine sodium salt). Chromatin was sonicated to an average size of ~250 bp using a Diagenode Bioruptor. ChIP was performed by using antibodies listed. Chromatin fromDrosophila(1:100 ratio to ESC or MN derived chromatin), andDrosophila-specific H2Av antibody were used as spike-in control in each sample.
Libraries were prepared according to manufacturer’s instructions (Illumina) as described in Narendra et al., 2015. Immunoprecipitated DNA was first end-repaired using End-It Repair Kit (Epicenter), tailed with an A using Klenow exo minus (NEB M0212), ligated to custom adapters, and fragments of 300±100 bp were size-selected. These fragments were amplified through ligation-mediated PCR amplification (LM-PCR) by using Q5 polymerase (NEB M0530).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq X |
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| Description |
WT ESCs
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| Data processing |
Sequence reads were mapped to mm10 reference genome with Bowtie 2.
After normalization with the spike-in Drosophila read counts for all (except for normalization with total counts for RAD21), normalized ChIP-seq read densities were visualized in Easeq Software and/or Integrative Genomics Viewer (IGV).
Assembly: mm10
Supplementary files format and content: bigwig
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| Submission date |
May 06, 2024 |
| Last update date |
Aug 21, 2024 |
| Contact name |
Danny Reinberg |
| Organization name |
University of Miami
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| Department |
Sylvester Cancer Center/Human Genetics
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| Lab |
Reinberg Lab
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| Street address |
1501 NW 10th Ave
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| City |
Miami |
| State/province |
Florida |
| ZIP/Postal code |
33136 |
| Country |
USA |
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| Platform ID |
GPL34328 |
| Series (2) |
| GSE230482 |
Members of an array of zinc finger proteins specify distinct Hox chromatin boundaries |
| GSE266783 |
Members of an array of zinc finger proteins specify distict Hox chromatin boundaries [ChIP-seq] |
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| Relations |
| BioSample |
SAMN41240316 |
| SRA |
SRX24476121 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8254496_RAD21_WT_E14_ESC_ns.bw |
280.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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