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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 08, 2018 |
| Title |
Fbxl19flfl_OHT_CDK8_rep1 |
| Sample type |
SRA |
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| Source name |
Fbxl19flfl_OHT_CDK8
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| Organism |
Mus musculus |
| Characteristics |
cell line: Fbxl19 fl/fl
cell type: mouse embryonic stem cells
treated with: 800nM 4-hydroxytamoxifen for 96 h
antibody: CDK8 (Bethyl Laboratories, A302-500A, lot A302-500A-1)
replicate: 1
passage: 13-20
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| Treatment protocol |
To generate Fbxl19ΔCXXC ES cells, cells were treated with 800nM 4-hydroxytamoxifen for 96 hours
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| Growth protocol |
Fbxl19fl/fl mouse ES cells were cultured on gelatine-coated dishes in DMEM (Thermo scientific) supplemented with 15% fetal bovine serum (BioSera), L-Glutamine, beta-mercaptoethanol, non-essential amino acids, penicillin/streptomycin (Thermo scientific) and 10ng/mL leukemia-inhibitory factor.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation was performed as described previously (Farcas et al., 2012) with slight modifications. Cells were fixed for 45 min with 2mM DSG (Thermo scientific) in PBS and 12.5 min with 1% formaldehyde (methanol-free, Thermo scientific). Reactions were quenched by the addition of glycine to a final concentration of 125µM. After cell lysis and chromatin extraction, chromatin was sonicated using a BioRuptor Pico sonicator (Diagenode), followed by centrifugation at 16,000×g for 20min at 4°C. 200µg chromatin diluted in ChIP dilution buffer (1% Triton-X100, 1mM EDTA, 20mM Tris-HCl (pH 8.0), 150mM NaCl) was used per IP. Chromatin was precleared with protein A Dynabeads blocked with 0.2 mg/ml BSA and 50 µg/ml yeast tRNA and incubated with the respective antibodies overnight at 4°C. Antibody-bound chromatin was purified using blocked protein A Dynabeads for 3 hours at 4°C. ChIP washes were performed as described previously (Farcas et al., 2012). ChIP DNA was eluted in ChIP elution buffer (1% SDS, 100mM NaHCO3) and reversed cross-linked overnight at 65°C with 200mM NaCl and RNase A (Sigma). The reverse cross-linked samples were treated with 20ug/ml Proteinase K and purified using ChIP DNA Clean&Concentrator™ kit (Zymo research).
ChIP DNA material was sheared with BioRuptor Pico sonicator to obtain DNA fragments of average size 200-800 bp. ChIPseq libraries were prepared with 30-40ng of ChIP DNA material using NEBNext Ultra DNA Library Prep Kit.
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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 |
Sequencing reads were aligned to the mouse genome (mm10) using bowtie2 (Langmead and Salzberg, 2012) the '-no-mixed' and '-no-discordant' options, and non-uniquely mapping reads were discarded . PCR duplicates were removed using samtools (Li et al., 2009).
Peak calling was performed using the MACS2 function with the “--broad” option using the biological replicates with matched input. Peak overlapping with a custom “blacklist” of artificially high genomic regions were discarded. Only peaks called in both replicates were considered.
Genome_build: mm10
Supplementary_files_format_and_content: Biological replicates were randomly downsampled to the same number of reads for each individual replicate and merged for visualisation. Bigwig files were generated using MACS2
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| Submission date |
May 10, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Emilia Dimitrova |
| E-mail(s) |
[email protected]
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| Organization name |
University of Oxford
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| Department |
Biochemistry
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| Lab |
Rob Klose
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| Street address |
South Parks Road
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| City |
Oxford |
| ZIP/Postal code |
OX1 3QU |
| Country |
United Kingdom |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE98754 |
FBXL19 transcriptionally primes promoters of developmental genes via CDK8-Mediator recruitment [ChIP-seq 2] |
| GSE98756 |
FBXL19 recruits CDK8-Mediator to CpG islands and primes developmental genes for activation during lineage commitment |
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| Relations |
| BioSample |
SAMN06925682 |
| SRA |
SRX2793944 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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