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| Status |
Public on Jan 21, 2020 |
| Title |
FBXL19fl_ES_TAM_rep2_CapC_set2 |
| Sample type |
SRA |
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| Source name |
mESCs_tamoxifen-treated (96hr OHT), CaptureC
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| Organism |
Mus musculus |
| Characteristics |
cell type: Mouse embryonic stem cells
cell line background: E14
cell line: Fbxl19fl
genotype: Fbxl19-CxxCfl/fl; Rosa26::ERT2-Cre
replicate: 2
treatment agent: tamoxifen (OHT)
treatment time point: 96 hr
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| Treatment protocol |
To induce conditional removal of the Fbxl19 CxxC domain or Med13 and Med13l in FBXL19fl and MED13/13lfl ESC lines, cells were treated with 800nM 4-hydroxytamoxifen (TAM) for 96 hours. To induce differentiation, 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. Human HEK293T cells used for calibrated ChIP-seq were grown at 37°C and 5% CO2, in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Labtech), 2 mM L-glutamine (Life Technologies), 1x penicillin-streptomycin solution (Life Technologies), and 0.5 mM beta-mercaptoethanol (Life Technologies). 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 |
CaptureC libraries were prepared as described previously (Hughes et al., Nature Genetics 2014). Briefly, 10 Million mouse ES cells were trypsinized, collected in 50ml falcon tubes in 9.3ml media and crosslinked with 1.25ml 16% formaldehyde for 10min at room temperature. Cells were quenched with 1.5M glycine, washed with PBS and lysed for 20 minutes at 4oC lysis buffer (10mM Tris pH 8, 10mM NaCl, 0.2% NP-40, supplemented with complete proteinase inhibitors) prior to snap freezing in 1ml lysis buffer at -80oC.
Lysates were then thawed on ice, pelleted and resuspended in 650µl 1x DpnII buffer (NEB). Three 1.5ml tubes with 200µl lysate each were treated in parallel with SDS (0.28% final concentration, 1hr, 37 oC, interval shaking 500rpm, 30sec on/30sec off), quenched with trypsin (1.67% final concentration, 1hr, 37 oC, interval shaking 500rpm, 30sec on/30sec off) and subjected to a 24 hour digestion with 3x10µl DpnII (homemade, 37 oC, interval shaking 500rpm, 30sec on/30sec off). Each chromatin aliquot was independently ligated with 8 µl T4 Ligase (240 U) in a volume of 1440µl (20 hours, 16oC). Following this, the nuclei containing ligated chromatin were pelleted to remove any non-nuclear chromatin, reverse-crosslinked and the ligated DNA was phenol-chloroform purified. The sample was resupended in 300µl water and sonicated 13x (Pico Bioruptor, 30sec on, 30sec off) or until a fragment size of approximately 200bp was reached. Fragments were size selected using AmpureX beads (Beckman Coulter: A63881, selection ratios: 0.85x / 0.4x). 2x 1-5µg of DNA were adaptor ligated and indexed using the NEBNext DNA library Prep Reagent Set (New England Biolabs: E6040S/L) and NEBNext Multiplex Oligos for Illumina Primer sets 1 (New England: E7335S/L) and 2 (New England: E7500S/L). The libraries were amplified 7x using Herculase II Fusion Polymerase kit (Agilent: 600677). Libraries were hybridized in teh following way: Probes were designed using the online tool by the Hughes lab (CapSequm: http://apps.molbiol.ox.ac.uk/CaptureC/cgi-bin/CapSequm.cgi) to be either 120bp (Sox2) or 70bp long each, two probes for each promoter of interest. The probes were pooled at a 2.9nM each and the samples were multiplexed by mass prior to hybridization. Hybridization was carried out using the Nimblegen SeqCap system (Roche, Nimblegen SeqCap EZ HE-oligo kit A # 06777287001, Nimblegen SeqCap EZ HE-oligo kit B # 06777317001, Nimblegen SeqCap EZ Accessory kit v2 # 07145594001, Nimblegen SeqCap EZ Hybridisation and wash kit # 05634261001) according to Roche protocol for 72 hours followed by a 24 hours hybridization (double Capture). The captured libraries were sequenced on Illumina NextSeq.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
processed data file:
Fli1_id3186524_FBXL19_ES_TAM_bg.bed_bg_sorted.bw
Mn1_id1964984_FBXL19_ES_TAM_bg.bed_bg_sorted.bw
Kif26b_id440003_FBXL19_ES_TAM_bg.bed_bg_sorted.bw
Dlk1_id4283906_FBXL19_ES_TAM_bg.bed_bg_sorted.bw
Spry1_id1010657_FBXL19_ES_TAM_bg.bed_bg_sorted.bw
Igdcc3_id3267532_FBXL19_ES_TAM_bg.bed_bg_sorted.bw
Col26a1_id2027715_FBXL19_ES_TAM_bg.bed_bg_sorted.bw
Samd14_id3955395_FBXL19_ES_TAM_bg.bed_bg_sorted.bw
Atoh8_id2237664_FBXL19_ES_TAM_bg.bed_bg_sorted.bw
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| Data processing |
library strategy: CaptureC
For CaptureC, paired-end reads were aligned and filtered for HiC artefacts using HiCUP (Wingett S, et al. (2015)) and Bowtie2 (Langmead and Salzberg, 2012) with fragment filter set to 100-800bp. Read counts of reads aligning to captured gene promoters and interaction scores (=significant interactions) were then called by CHiCAGO (Cairns et al., 2016).
For visualisation of CaptureC data weighted pooled read counts from chicago data files were normalized to total read count aligning to captured gene promoters in the sample and further to the number of promoters in the respective capture experiment. Bigwig files were generated from these normalized read counts.
For comparative boxplot analysis interactions called by chicago (score >= 5) across all samples were aggregated and interactions with a distance of less than 4 DpnII fragments were merged to a single interaction peak. For each interaction peak we then quantified mean normalized read count and chicago score of all overlapping DpnII fragments.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files showing the genome coverage of normalized read counts for each sample and each promoter were generated from chicago data object.
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| Submission date |
Aug 28, 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 |
GPL19057 |
| Series (2) |
| GSE136423 |
CDK-Mediator and FBXL19 cooperate in the induction of developmental genes by promoting regulatory interactions [CaptureC] |
| GSE136424 |
CDK-Mediator and FBXL19 cooperate in the induction of developmental genes by promoting regulatory interactions |
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| Relations |
| BioSample |
SAMN12647297 |
| SRA |
SRX6769629 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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