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| Status |
Public on May 17, 2024 |
| Title |
3rG4 cl5 k27me3 rep2 input |
| Sample type |
SRA |
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| Source name |
Day 7 differentiated mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
strain: 129S1/CastEiJ x 129S1
cell type: Day 7 differentiated mouse embryonic stem cells
genotype: Xist 3'rG4 mutant
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| Treatment protocol |
Cells were cross-linked in PBS with 1% formaldehyde at room temp for 20 min with gentle agitation and quenched with 0.125 M glycine for 10 min. Fixed cells were washed twice with PBS, harvested by scraping and stored at -80 °C until use.
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| Growth protocol |
Clonal TsixTST/+ mESC (TST) line was cultured as routine procedure. mESC were grown on inactivated feeders in high glucose D-MEM supplemented with 15% Hyclone FBS, 25 mM HEPES pH 7.2-7.5, 1x MEM non-essential amino acids, 1x Pen/Strep, 0.1 mM βME, and 500 U/mL ESGRO recombinant mouse Leukemia Inhibitory Factor (LIF) protein at 37°C with 5% CO2. EB differentiation was performed with growing feeder-depleted cells in the medium without LIF. Cells were kept in suspension until 3 days of differentiation and transferred to gelatinized plates on day 3 for further adherent growth.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
10 million cells per ChIP were resuspended with 0.5 ml ice-cold ChIP lysis buffer (50 mM Tris-HCl pH8, 10 mM EDTA pH8, 1% SDS). Cell extracts were subjected to sonication using the Qsonica apparatus (40% power, 30s on/ 30s off for total sonication time 30 min at 4°C). Sheared chromatin was checked on the 1% agarose gel electrophoresis, repeat sonication a second time if the fragment is larger than 1 kb marker. 0.1 ml lysate was mixed with 1.2 ml ChIP dilution buffer (20 mM Tris-HCl pH8, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA pH8), and incubated with 2 μg antibody for overnight at 4°C. Immunoprotein complex was captured with 20 μL Dynabeads Protein G for 5 hr incubation. Afterward, beads were washed three times with ChIP wash buffer 1 (20 mM Tris-HCl pH8, 150 mM NaCl, 2mM EDTA pH8, 1% Triton X-100, 0.1% SDS) and once with ChIP wash buffer 2 (20 mM Tris-HCl pH8, 500 mM NaCl, 2mM EDTA pH8, 1% Triton X-100, 0.1% SDS). 100 μL of ChIP elution buffer (1% SDS, 100 mM NaHCO3, 40 ug/ml RNaseA) was added to the washed bead and incubated for 1hr at 37°C to recover bound nucleoproteins, dynabeads were removed by using magnetic stand. Reverse-crosslinking was done by incubating the supernatant at 65° C overnight.
NEBNext Ultra II DNA Kit (NEB) was used for library preparation by following the product manual.
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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 |
Adaptor sequences and PCR duplicates are removed from the raw read files. For Xi (mus) and Xa (cas) allelic analysis, adaptor trimmed total sequencing reads were allele-specifically aligned to 129S1/SvJm (mus) and CAST/Eih (cas) genomes. FPM and input normalized BigWig track files are generated for visualization and further quantification of H3K27me3 density on the different gene groups. mm10 bigwig files were created using the UCSC tool LiftOver from mm9 aligned bigwig files
Assembly: mm9
Supplementary files format and content: bigWig
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| Submission date |
Dec 05, 2023 |
| Last update date |
May 17, 2024 |
| Contact name |
yongwoo lee |
| E-mail(s) |
ylee@molbio.mgh.harvard.edu
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| Phone |
6177552856
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| Organization name |
MASSACHUSETTS GENERAL HOSPITAL
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| Department |
Mol bio
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| Lab |
Jeannie Lee Lab
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| Street address |
185 Cambridge St
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE219082 |
Epigenetic regulation by dynamic RNA G-quadruplex folding and unfolding (ChIP-Seq) |
| GSE219083 |
G-quadruplex folding in Xist RNA antagonizes PRC2 activity for step-wise regulation of X-chromosome inactivation |
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| Relations |
| BioSample |
SAMN38675615 |
| SRA |
SRX22779230 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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