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| Status |
Public on May 31, 2021 |
| Title |
RING1B_ERT2-Cre_Pcgf1Control_ES_rep2 |
| Sample type |
SRA |
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| Source name |
ES
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| Organism |
Mus musculus |
| Characteristics |
tamoxifen treatment: without 4-OHT
genotype: ERT2-Cre;Pcgf1f/f
cell type: embryonic stem cell
antibody: RING1B (Laboratory made)
replicate: 2
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| Growth protocol |
Embryonic stem cells (ESCs) were cultured with LIF (1U: Laboratory made), SU5402 (Altair corporation), PD184352 (Altair corporation) and CHIR99021 (Altair corporation) on MEF. For Embryoid Body (EB) formation, ESCs were cultured without LIF, SU5402, PD184352, CHIR99021 for 2 days on low attach culture dish. For epiblast stem cell (EpiSC) establishment, Embryoid Body (day2) are dispersed. And the cells were cultured on MEF with ActivinA (Peprotech), bFGF (Wako) and IWP-2 (Wako) on MEF in F12 medium (Gibco) 1%KSR (Gibco).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
10^6-10^7 cells were cross-linking by 1% formaldehyde for 10 min at room temperature. Cross-linked cells were washed once with PBS, added swelling buffer (0.1% NP-40, 1 mM DTT in PBS) and chilled on ice for 10 minutes. After centrifuge, pellets were suspended with RIPA buffer (10 mM Tris-HCl pH8.0, 1mM EDTA, 140 mM NaCl, 1%Triton X-100, 0.1% SDS, 0.1% Sodium Deoxycholate), and then were sonicated by using a BioRuptor sonicator (Diagenode). After sonication, DNA concentration is measured with Qubit 1x dsDNA HS Assay Kit (Thermo, cat. Q33230). On the other hand, protein A/G beads were prepared as washed twice with beads blocking buffer and suspended with beads blocking buffer with approximately 5 µg of antibody and incubated at 4ºC for more than 2 hours. Then, antibody attached beads were washed twice with beads blocking buffer and the chromatin samples were added to those antibody-attached-beads. After overnight incubation at 4ºC, chromatin samples bound beads were washed with RIPA for 6 times, RIPA high salt buffer(10 mM Tris-HCl pH8.0, 1mM EDTA, 500 mM NaCl, 1%Triton X-100, 0.1% SDS, 0.1% Sodium Deoxycholate) twice, LiCl buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Sodium Deoxycholate) twice and TE twice. The chromatins were eluted with elution buffer (10 mM Tris-HCl pH8.0, 5 mM EDTA, 300 mM NaCl, 0.1 % SDS) overnight at 65ºC and treated with RNase for 30 min for 37°C and further incubation with Proteinase K for 1 hour at 37ºC. Then DNA samples were purified and used for further ChIP-qPCR or ChIP-seq.
ChIP-seq libraries were prepared by using NEBNext Ultra II FS DNA prep kit (NEB E7805) following manufacturer’s instruction.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Sequencing was performed with 75 bp single-end read by NextSeq500 (run by Kazusa DNA Research Institute) or 50bp single-end read by HiSeq1500 (run in RIKEN-IMS NGS facility).
The data were mapped onto UCSC-mm9 from illumina igenome. Generated sam format file combine to bam format file using samtools view (version 1.3.1). Then, PCR duplicate and multiple aligned reads are removed.
For bigwig generation, bam format files were processed by deeptools bamCoverage. The data of CpG island and TSS are derived from UCSC mm9.
Genome_build: mm9
Supplementary_files_format_and_content: bigWig
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| Submission date |
Dec 04, 2019 |
| Last update date |
May 31, 2021 |
| Contact name |
Hiroki Sugishita |
| E-mail(s) |
hiroki.sugishita@riken.jp
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| Organization name |
RIKEN
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| Department |
IMS
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| Lab |
Laboratory for Developmental Genetics
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| Street address |
1-7-22, Suehiro-cho
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| City |
Tsurumi-ku,Yokohama |
| State/province |
Kanagawa |
| ZIP/Postal code |
230-0045 |
| Country |
Japan |
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| Platform ID |
GPL18480 |
| Series (2) |
| GSE141485 |
Variant PCGF1-PRC1 links PRC2 recruitment with induced transcriptional inactivation at target genes [ChIP-seq] |
| GSE141488 |
Variant PCGF1-PRC1 links PRC2 recruitment with induced transcriptional inactivation at target genes |
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| Relations |
| BioSample |
SAMN13482145 |
| SRA |
SRX7268236 |
| Supplementary data files not provided |
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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