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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 31, 2016 |
| Title |
Input NRC ZT 14 Bmal1 KO |
| Sample type |
SRA |
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| Source name |
Liver, Bmal1 KO, ZT 14, input
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| Organism |
Mus musculus |
| Characteristics |
strain/background: C57/BL6
genotype/variation: Bmal1 KO
gender: male
feeding: night-restricted feeding
tissue: liver
time point: ZT 14
chip antibody: none
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| Growth protocol |
C57/BL6 male, 12- to 14-wk-old (at time of sacrifice) mice were housed in a 12 h light/12 h dark (LD) regimen for 2 wk with water and food available ad libitum. They were then phase-entrained to a 12 h/12 h LD regimen with water ad libitum but food access between ZT12 and ZT24 for 7 d (ZT, Zeitgeber time; ZT0 is defined as the time when the lights are turned on and ZT12 as the time when lights are turned off). At each ZT2, ZT06, ZT10, ZT14, ZT18, ZT22, and ZT26, five mice were anesthetized with isoflurane and decapitated. The livers were perfused with 2 ml of PBS through the spleen and immediately collected. A small piece of liver tissue (approx. 100 mg) was snap-frozen in liquid nitrogen and kept at -80°C for RNA extraction. The remaining liver tissue was immediately homogenized in PBS containing 1% formaldehyde for chromatin preparation. All animal care and handling was performed according to the State of Geneva's law for animal protection.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNase1-seq protocol: Mouse liver nuclei were prepared as described in (Tian and Schibler 1991). Freshly prepared nuclei were suspended in ice-cold Ψ-buffer (11 mM KPO4 pH 7.4, 108 mM KCl, 22 mM NaCl, 5mM MgCl, 1 mM CaCl2, 1 mM DTT) and pelleted. 5x10^6 nuclei were suspended in 200 μl of Ψ-buffer supplemented with 0.2% of NP40 and 1 u/ml of DNaseI (DPFF Worthington Biochemical Corporation). DNase1 digestion was performed for 6 minutes in room temperature and the reaction was stopped by adding 200 μl of lysis buffer (50mM Tris-HCl pH 8, 20 mM EDTA, 1% SDS, 200 μg/ml proteinaseK). Protease digestion was performed overnight at 55 oC. RNaseA (100 μg/ml) was then added and samples were incubated at 37 oC for an hour. DNA was then extracted twice with phenol-chloroform and precipitated with isopropanol in the presence of 0.5 M NaCl. DNAs were dissolved in 5 mM Tris-HCl pH 8. DNAs from 4 animals were pooled and 75 μg of DNA was loaded on 11 ml 10%-50% sucrose gradient in STE buffer (1M NaCl, 20 mM Tris-HCl pH 8, 5 mM EDTA) and centrifuged at 30000 rpm for 16 hours at 20 oC (SW 40 Ti rotor, Beckman Coulter Inc). The sucrose gradients were then fractionated and DNA was precipitated by two volumes of ethanol in the presence of 5 μg of glycogen. Fractions containing DNA sized around 300bp were pooled and used for Illumina library preparation.
ChIP-seq PolII protocol: Perfused livers were processed for chromatin preparation as described in Ripperger JA, Schibler U (2006). The chromatin samples from the five mice were then pooled, frozen in liquid nitrogen, and stored at -80°C. For the ChIP experiments, the following antibody was used: anti-RPB2 (Santa Cruz Biotechnology, sc-673-18). To determine the optimal amounts of antibody, we performed pilot ChIP assays and determined the enrichment for a set of promoters by real-time qPCR according to Ripperger JA, Schibler U (2006). A total of 1 ml of each chromatin suspension (containing about 60 µg of DNA) was incubated with 10 µg of anti-RPB2 in buffer A (20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA) overnight at 4°C on a rotating wheel. Ten µl of protein A bead suspension (25% slurry in buffer A), pre-blocked with 10 µg/ml of salmon sperm DNA and BSA at 4°C overnight, was then added and the incubation was continued for 1 h at room temperature on a rotating wheel. The beads were then washed with dialysis buffer and ChIP wash buffer as described in O'Geen H, Nicolet CM, Blahnik K, Green R, Farnham PJ (2006). Protein-DNA complexes were eluted from the beads, de-cross-linked, and treated with RNase A and, subsequently, with proteinase K, as described in O'Geen H, Nicolet CM, Blahnik K, Green R, Farnham PJ (2006). The DNA concentration was determined by fluorometry on the Qubit system (Invitrogen). A total of 10-12 ng DNA were used for the preparation of the library.
ChIP-seq H3K27ac protocol: ChIPs were performed according to the method described by Reddy et al. (2009) with a few modifications. The 100 ul chromatin aliquots were used for each IP and diluted with 900 ul of RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS at pH 7.4) and added to dynal magnetic beads conjugated with (sheep anti-mouse IgG Dynabeads, Invitrogen, Cat no: 110-31) pre-treated with 3 ul of polyclonal antibody for H3K27ac (Active Motif, Cat no: 39135) for immunoprecipitation of specific complexes. The samples were incubated overnight at 4°C on rotator, then magnetic beads washed 7 times with lithiumchloride wash buffer (100mM Tris at pH 7.5, 500mM LiCl, 1% NP-40 and 1% sodiumdeoxycholate) and finally once with 1X TE buffer (10mM Tris-HCl at pH 7.5, 0.1mM Na2EDTA). The chromatin complex was eluted using elution buffer (1% SDS, 0.1M NaHCO3) for 1 h at 65°C using eppendorf thermo-mixer. The chromatin was then de-crosslinked overnight at 65°C and ChIP DNA purified using Qiagen PCR purification kit and eluted in 50 ul of elution buffer. For qPCR reaction, 1.5 ul of 1/10 diluted ChIP DNA is used.
Libraries for ultra-high-throughput sequencing were prepared with the ChIP-Seq DNA sample kit (Illumina) as recommended by the manufacturer.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Chromatin
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| Data processing |
All alignments were performed using the BBCF HTSstation (available at http://htsstation.epfl.ch , David et al., 2014).
Peak calling was done using ChIP-peak (Schmid CD, Bucher P. 2010, Ambrosini G., Dreos R. and Bucher P. IWBBIO 2014, Available: http://ccg.vital-it.ch/chipseq/chip_peak.php) on DNase signal using all ZT concatenated with the following parameters: cutoff=100,vicinity=400,window size=600, threshold=1000. A peak calling was done as well specifically for ZT6 in the WT and in the Bmal1KO context with the following parameters: cutoff=100,vicinity=1000,window size=600, threshold=200. Detected peaks were quantified using the DNase and PolII signal with a window of +/-300 bp around peak center and using the H3K27ac with a window of +/-1kb peak center for each time point and in each context.
Wig files were generated using bam2wig (see David et al., 2014) and were normalized according to the number mapped reads divided by 10e7. DNase I signal is represented using the first position of the reads considered as the cutting position and without strand shifting. Pol II and H3K27ac are represented using the whole read length and a shift of respectively 80bp and 90bp for PolII and H3K27ac.
DNase hypersensitive sites footprints were detected using Wellington algorithm (Piper et al. 2013) with the following parameters: -sh 20,36,5 -fdr 0.05
Genome_build: MGSCv37 (mm9)
Supplementary_files_format_and_content: Processed data files contain the normalized signal for each mark at each time point in BigWig format. In addition, the DNaseI hypersensitive peaks detected by the peak calling tool are provided in BigBed format. The footprints detected by Wellington algorithm are provided in BigWig format where the footprint score is reproted in negative scale (see Piper 2013). A table of quantile-normalized quantification of each mark at each time point and in WT and Bmal1KO context is provided in text format.
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| Submission date |
Sep 29, 2014 |
| Last update date |
May 04, 2020 |
| Contact name |
Jonathan Aryeh Sobel |
| E-mail(s) |
jonathan.sobel@epfl.ch
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| Organization name |
EPFL
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| Department |
School of Life Sciences
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| Lab |
Felix Naef Lab
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| Street address |
EPFL SV IBI-SV UPNAE AAB 0 36 (Batiment AAB) Station 15
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| City |
Lausanne |
| State/province |
Vaud |
| ZIP/Postal code |
CH-1015 |
| Country |
Switzerland |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE60430 |
Regulatory logic of the coupled diurnal and feeding cycles in the mouse liver [DNase-seq, ChIP-seq] |
| GSE60578 |
Regulatory logic of the coupled diurnal and feeding cycles in the mouse liver |
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| Relations |
| BioSample |
SAMN03084072 |
| SRA |
SRX716885 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not applicable for this record |
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