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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 08, 2018 |
| Title |
Fbxl19fl_72hrTAM_rep2 |
| Sample type |
SRA |
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| Source name |
Fbxl19fl_72hrTAM
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| Organism |
Mus musculus |
| Characteristics |
cell line: Fbxl19fl/fl
cell type: mouse embryonic stem cells
replicate: 2
passage: 13-20
treated with: 800nM 4-hydroxytamoxifen for 72 h
differentiation: no
molecule subtype: nascent RNA
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| Treatment protocol |
Fbxl19fl/fl ES cells were treated with 800nM 4-hydroxytamoxifen (Sigma) for 72 hours in order to delete the CxxC domain. For differentiation of ES cells, 4x10^4 cells/cm2 were allowed to attached to gelatinised dishes (~12 hours) and treated with 1µM retinoic acid (Sigma-Aldrich) in EC-10 medium (DMEM supplemented with 10% fetal bovine serum, L-Glutamine, beta-mercaptoethanol, non-essential amino acids and penicillin/streptomycin) for 72 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 |
total RNA |
| Extraction protocol |
For labelling of nascent RNA transcripts, cells were grown in 15cm culture dishes. Labelling was performed for 20 minutes at 37°C by the addition of 50mM 4-thiouridine (T4509, Sigma) to the culture medium. After the incubation, the medium was removed and RNA was isolated using TRIzol reagent (Thermo scientific) following manufacturer’s instructions. The total RNA was treated with TURBO DNA-free kit (Ambion, Thermo scientific) in order to remove contaminating genomic DNA. 300µg RNA was biotinylated using 600 µg Biotin-HPDP (21341, Pierce, Thermo scientific) in Biotinylation buffer (100mM Tris-HCl pH 7.4, 10mM EDTA). The reaction was carried out for one and half hour on a rotor at room temperature. Unincorporated Biotin-HPDP was removed by chloroform/isoamylalcohol (24:1, Sigma) wash followed by isopropanol precipitation of the biotinylated RNA. Labelled biotinylated RNA was isolated using µMacs Streptavidin Kit (130-074-101, Miltenyi) following manufacturer’s instructions and purified using RNeasy MinElute Cleanup kit (Qiagen). The quality of the RNA was confirmed using the RNA pico Bioanalyser kit (Agilent).
Up to 1µg purified 4sU-labelled RNA was used to prepare libraries for 4sU-seq. Ribosomal RNA was removed using NEBNext® rRNA Depletion Kit and libraries were prepared using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB) following manufacturer’s instructions. Library quality was assessed using the High-sensitivity DS DNA Bioanalyser kit (Agilent) and the concentration was measured by quantitative PCR using KAPA Library quantification standards for Illumina (KAPA Bioasystems). All RNA-seq experiments were carried out in biological triplicates. RNA-seq libraries were sequenced as 76 paired-end reads on Illumina NextSeq500 platform using NextSeq® 500/550 High Output Kit v2 (150 cycles).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Fbxl19ΔCxxC
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| Data processing |
rRNA reads were initially removed by aligning the data to mouse rRNA genomic sequences (GenBank: BK000964.3) using bowtie2. The rRNA-depleted reads were next aligned against mm10 genome using the STAR RNA-seq aligner (Dobin et al., 2012). To improve mapping of nascent, intronic sequences, a second alignment step with bowtie2 was included using the reads which failed to map using STAR. PCR duplicates were removed using samtools (Li et al., 2009).
Genome_build: mm10
Supplementary_files_format_and_content: Stranded bigWig files representing genome coverage for merged biological triplicate.
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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) |
| GSE98755 |
FBXL19 transcriptionally primes promoters of developmental genes via CDK8-Mediator recruitment [RNA-seq] |
| GSE98756 |
FBXL19 recruits CDK8-Mediator to CpG islands and primes developmental genes for activation during lineage commitment |
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| Relations |
| BioSample |
SAMN06925685 |
| SRA |
SRX2793984 |
| 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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