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| Status |
Public on Jan 21, 2020 |
| Title |
FBXL19fl_RA_UNT_rep1_NatInput |
| Sample type |
SRA |
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| Source name |
mESCs_control, retinoic-acid treated (72hr RA)_input
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| Organisms |
Drosophila melanogaster; Mus musculus |
| Characteristics |
cell type: Mouse embryonic stem cells (mESCs)
genotype/vairation: Fbxl19fl; inducible conditional removal of FBXL19 CxxC domain
replicate: 1
treatment agent: retinoic acid (RA)
treatment time point: 72 hr
spike-in reference organism: Drosophila melanogaster
spike-in cell line: SG4
chip antibody: none
chip antibody info.: none
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| Treatment protocol |
To induce conditional removal of FBXL19 CxxC domain, cells were treated with 800 nM 4-hydroxytamoxifen (TAM) for 96 hours. To induce RA-differentiation mouse ES cells were treated with 1uM retinoic acid (RA) for 72 hours.
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| Growth protocol |
Mouse embryonic stem cells were grown on gelatin-coated plates at 37°C and 5% CO2, in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% fetal bovine serum (Labtech), 2 mM L-glutamine (Life Technologies), 1x penicillin-streptomycin solution (Life Technologies), 1x non-essential amino acids (Life Technologies), 0.5 mM beta-mercaptoethanol (Life Technologies), and 10 ng/mL leukemia inhibitory factor. Drosophila S2 (SG4) cells were grown adhesively at 25°C in Schneider’s Drosophila Medium (Life Technologies), supplemented with 1x penicillin-streptomycin solution (Life Technologies) and 10% heat-inactivated fetal bovine serum (Labtech).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For native cChIP-seq, 5 x 10^7 mouse ESCs (both untreated and following tamoxifen treatment) were mixed with 2 x 10^7 Drosophila SG4 cells in PBS. Mixed cells were pelleted and nuclei were released by resuspending in ice cold lysis buffer (10mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 5 mM N-ethylmaleimide). Nuclei were then washed, and resuspended in 1 ml of MNase digestion buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.25M sucrose, 3mM CaCl2, 10 mM N-ethylmaleimide, 1x protease inhibitors (Sigma)). Each sample was then incubated with 200 units of MNase (Fermentas) at 37°C for 5 min, followed by the addition of 4 mM EDTA to halt MNase digestion. Following centrifugation at 1500 g for 5 min at 4°C, the supernatant (S1) was retained. The remaining pellet was incubated with 300 µl of nucleosome release buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2 mM EDTA, 1x protease inhibitors, 10 mM N-ethylmaleimide) at 4°C for 1 h, passed five times through a 27G needle using a 1 ml syringe, and spun at 1500 g for 5 min at 4°C. The second supernatant (S2) was collected and combined with corresponding S1 sample from above. A small amount of S1/S2 DNA was purified and visualized on a 1.5% agarose gel to confirm digestion to mostly mono-nucleosomes.
Libraries for both ChIP and Input samples were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina, following manufacturer’s guidelines. Samples were indexed using NEBNext Multiplex Oligos. The average size and concentration of all libraries was analysed using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent) followed by qPCR using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche). Libraries were sequenced as 40 bp paired-end reads on Illumina NextSeq 500 platform in biological duplicate.
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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 |
Paired-end reads were aligned to the genome sequence of concatenated mouse and spike-in genomes (mm10+dm6) using Bowtie 2 with the “--no-mixed” and “--no-discordant” options specified. Reads that were mapped more than once were discarded, followed by removal of PCR duplicates with SAMTools for native cChIP-seq or Sambamba for cross-linked cChIP-seq.
To internally calibrated cChIP-seq experiments, we spiked-in a fixed number of control cells (Drosophila SG4 cells) to each experimental sample. For annotation of genomic intervals and data visualisation, mm10 reads were randomly subsampled using factors that reflected the total number of dm6 reads in each sample. To account for any variation in spike-in cell mixing in different biological replicates, the downsampling factors were additionally corrected using the ratio of dm6/mm10 total read counts in corresponding Input samples.
For each condition, biological replicates were merged for downstream applications. Genome coverage tracks were generated using the pileup function from MACS2 (Zhang et al. 2008).
Metaprofile visualization was carried out using the Deeptools (Ramirez et al., 2016) software and the commands i) computeMatrix reference-point with the option --referencePoint center and ii) plotHeatmap.
ChIP-seq enrichments were quantified from bedgraph files using the annotatePeaks.pl script from HOMER (Heinz et al., 2010). Log2 fold changes between samples were calculated following the addition of a pseudocount of 1.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files representing genome coverage for merged replicates of spike-in normalised cChIP-seq profiles
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| Submission date |
Aug 27, 2019 |
| Last update date |
Jan 21, 2020 |
| Contact name |
Angelika Feldmann |
| E-mail(s) |
[email protected]
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| Phone |
7983705260
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| Organization name |
University of Oxford, Department of Biochemistry
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| Street address |
South Parks Road
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| City |
Oxford |
| State/province |
England |
| ZIP/Postal code |
OX1 3QU |
| Country |
United Kingdom |
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| Platform ID |
GPL25537 |
| Series (2) |
| GSE136422 |
CDK-Mediator and FBXL19 cooperate in the induction of developmental genes by promoting regulatory interactions [ChIP-seq] |
| GSE136424 |
CDK-Mediator and FBXL19 cooperate in the induction of developmental genes by promoting regulatory interactions |
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| Relations |
| BioSample |
SAMN12642765 |
| SRA |
SRX6762293 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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