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| Status |
Public on Jan 31, 2024 |
| Title |
TMRM_H3K27me3_high_1 |
| 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
chip antibody: H3K27me3 (Millipore, #07-449)
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| Growth protocol |
Naive ESCs were maintained in 2i/LIF culture condition, which contains N2B27 basal medium supplemented with PD0325901 (1 µM), CHIR99021 (3 µM) and LIF (1000 U/ml). N2B27 consists of a 1:1 mix of DMEM/F12 and Neurobasal, N2 (1:100), B27 (1:100), 2 mM Glutamine, Penicillin-Streptomycin (1:100) and 0.055 mM 2-mercaptoethanol.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were sorted and resuspended in the wash buffer (20 mM HEPES pH7.5, 150 mM NaCl, 0.5 mM spermidine, 1X protease inhibitor cocktail). 10 µl of Concanavalin A-coated beads (Bangs Laboratories, Inc., #BP531) were added into the cell suspension with rotation for 10 min.
Bead-bound cells were then incubated overnight at 4oC with Rabbit anti-H3K4me3 (Millipore, #07-473) or Rabbit anti-H3K27me3 (Millipore, #07-449) antibody diluted in the antibody buffer (EDTA/Digitonin/wash buffer) (1 µg antibody in 500 µl antibody buffer). The beads were then washed three times with the wash buffer containing Digitonin (EMD Millipore, #300410), followed by an incubation with 200 µl pA-MNase (0.7 ng/µl) for 1 hour at 4oC. Next, the beads were washed three times with the wash buffer containing Digitonin before 2 mM of CaCl2 were added to active the pA-MNase for 30 min on ice. The reaction was quenched by 2X stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% Digitonin, 50 µg/ml RNase A (QIAGEN, #19101), 50 µg/ml glycogen (Roche, #10901393001) and 2 pg/ml Drosophila spike-in DNA) at 37oC for 10 min. The digested target chromatin was released and purified by phenol, chloroform, and ethanol. Libraries were prepared with the NEBNext UltraTM II DNA library Prep kit (NEB, #E7645L) and 10 cycles PCR.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
High mitochondrial membrane potential (Δ
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| Data processing |
For H3K4me3 and H3K27me3 CUT&RUN data, paired-end raw sequencing reads were trimmed with Trim Galore to remove adaptors and low-quality reads.
Processed read (>20 bp) were mapped back to the mouse mm10 reference genome using bowtie2 (v2.4.2) with the following parameters “--local --very-sensitive-local --no-unal --no-mixed --no-discordant -q --phred33 -I 10 -X 700 --threads 12’.
For H3K4me3, filtered BAM files for each sample were submitted to the callpeak function of MACS2 (v2.2.7.1) for peak regions calling with parameters “-p 1e-5 -g mm”.
For H3K27me3 domains, peak calling was performed using SICER (v1.1) with default parameters for calling broad peaks.
Assembly: mm10 (GRCm38)
Supplementary files format and content: bed files (peaks called for H3K27me3 and H3K4me3)
Library strategy: CUT&RUN
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| Submission date |
Aug 10, 2022 |
| Last update date |
Jan 31, 2024 |
| Contact name |
Wentao Yang |
| E-mail(s) |
wentaoy@usc.edu
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| Organization name |
university of southern california
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| Street address |
1441 Eastlake Ave
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| City |
Los Angeles |
| State/province |
California |
| ZIP/Postal code |
90033 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE210913 |
Quiescence Enables Unrestricted Cell Fate in Naive Embryonic Stem Cells |
| GSE210915 |
Quiescence Enables Unrestricted Cell Fate in Naive Embryonic Stem Cells |
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| Relations |
| BioSample |
SAMN30237405 |
| SRA |
SRX17020207 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6442914_H3K27me3sicerhigh.rep1.bed.gz |
338.3 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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