 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 01, 2013 |
| Title |
CREB ChIP-seq in Fasted Liver (Rep4) |
| Sample type |
SRA |
| |
|
| Source name |
mouse whole liver
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57/Bl6
tissue: liver
condition: Fasted 24h
time of day: ZT03
target: CREB
antibody: CREB-1 Antibody (Santa Cruz Biotech, catalog# sc-186)
|
| Treatment protocol |
Fasted mice had food removed from their cages for 24h prior to liver harvest. Re-fed mice had food removed from their cages for 24h, and then were given access to food for 2h prior to liver harvest. Ad-lib fed mice were given unrestricted access to food.
|
| Growth protocol |
All mice used in this study were 8-12 week-old male C57BL/6J mice.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin extraction: Mouse livers were minced finely in cold phosphate-buffered saline (PBS) and cross-linked in 1% formaldehyde for 10 min while rotating. Cross-linking was quenched by adding glycine to a final concentration of 0.125 M for 5 min while rotating. The tissue was rinsed in cold PBS and homogenized with a Dounce homogenizer in cold cell lysis buffer (10 mM Tris–Cl, pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40) and protease inhibitors. Cells were incubated at 4°C for 5 min to release nuclei. Nuclei were centrifuged at 13 000 g for 5 min to form a pellet. The pellet was resuspended in nuclear lysis buffer [1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 50 mM Tris–Cl, pH 8.1) and protease inhibitors and sonicated using the Diagenode Bioruptor for 10 min on high, using 30 s intervals. Debris were removed by centrifugation at 13 000 g for 10 min, and the supernatant was collected and snap frozen in liquid nitrogen. A 10 μl aliquot was reversed by the addition of NaCl to a final concentration of 192 mM, overnight incubation at 65°C, and purification using a PCR purification kit (Qiagen, CA, USA). The chromatin concentration was determined using a NanoDrop 3.1.0 nucleic acid assay (Agilent Technologies, Santa Clara, CA, USA).
Chromatin Immunoprecipitation: Ten micrograms of chromatin per sample was precleared by adding 90 μl of protein A/G-agarose in 1 ml of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 167 mM NaCl, 16.7 mM Tris–Cl, pH 8.1) and rotating the sample for 1 h at 4°C. Protein A/G-agarose was sedimented by centrifugation at 3000 g for 30 s. Two micrograms of antibody was added to the supernatant and incubated overnight at 4°C. Protein A/G-agarose was blocked overnight at 4°C with 1 mg/ml bovine serum albumin in ChIP dilution buffer, added to the chromatin, and rotated for 1 h at 4°C. Following three consecutive washes of 5 min each with TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–Cl, pH 8.1, 150 mM NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–Cl, pH 8.1, 500 mM NaCl) and ChIP buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris–Cl, pH 8.1), chromatin was eluted by adding 100 μl of freshly made ChIP elution buffer (1% SDS, 0.1 M NHCO3) to the pellet and rotating the sample for 10 min. Elution was repeated with an additional 100 μl of ChIP elution buffer, and the eluates were combined. Cross-linking was reversed by the addition of NaCl to a final concentration of 192 mM and overnight incubation at 65°C.
Immunoprecipitated DNA was modified for sequencing following the manufacturer’s protocol (Illumina). Briefly, DNA samples were blunted with a combination of T4 DNA polymerase, Klenow polymerase, and T4 PNK, then a single 3′-end “A” base was added using Klenow exo (3′ to 5′ exo minus). Adapters provided by Illumina were then ligated to the ends of the modified DNA before size selection of ~200bp fragments via PAGE extraction. The isolated DNA was then amplified by 18 cycles of PCR as described. PCR products were column purified with the QIAquick PCR Purification Kit (Qiagen) and assayed for quantity and quality with the Agilent 2100 Bioanalyzer (Agilent Technologies), then diluted to a concentration of 10nM. All sequencing was performed on an Illumina GAIIx, some libraries were sequenced twice to achieve sufficient read counts.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
CREB_Fasted_Liver_4
CREB_peaks_fasted_final.bed
|
| Data processing |
Mapping was performed using the Illumina Eland pipeline and the mm8 reference genome.
Mapped reads were filtered to discard ambiguously aligned reads and reads mapping with greater than 2 mismatches. When multiple reads in a biological replicate mapped to an identical genomic position (redundant reads), only one such read was used for further processing.
CREB Peak Calling: For each condition (fasted, re-fed), non-redundant reads from all five biological replicates were pooled into a single read set, and initial peak-calling was performed with HOMER v3.0 (5% FDR), using a large pool of previous experimental inputs as the background control. To identify peaks that are not dependent on any single replicate, additional read subsets were created by pooling every possible combination of four replicates from each condition (fasted or re-fed), and peak-calling was repeated with the same parameters. Initial peak-calls from the 5-replicate pool were discarded if they failed to be called in any of the 4-replicate pools. Peaks passing this "Single-Sample Independence" test were further filtered by removing all peaks with height less than 0.35 reads per million, which was an empirical cut-off determined by validation of randomly selected peaks.
Genome_build: MGSCv36
Supplementary_files_format_and_content: BedGraph: Stack height profiles of each ChIP-seq biological replicate are provided in BedGraph format. Non-redundant reads were extended to 108bp fragments before computing stack height profiles.
Supplementary_files_format_and_content: BED: CREB peak calls are provided in BED format for each condition, with peak height normalized to reads per million in the score column.
|
| |
|
| Submission date |
Apr 01, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Logan J Everett |
| Organization name |
U.S. Environmental Protection Agency
|
| Department |
Office of Research and Development
|
| Lab |
Center for Computational Toxicology and Exposure
|
| Street address |
109 T.W. Alexander Dr
|
| City |
RTP |
| State/province |
North Carolina |
| ZIP/Postal code |
27711 |
| Country |
USA |
| |
|
| Platform ID |
GPL11002 |
| Series (2) |
| GSE45674 |
Integrative genomic analysis of CREB defines a critical role for transcription factor networks in mediating the fed/fasted switch in liver [ChIP-seq] |
| GSE45733 |
Integrative genomic analysis of CREB defines a critical role for transcription factor networks in mediating the fed/fasted switch in liver |
|
| Relations |
| SRA |
SRX257616 |
| BioSample |
SAMN01995749 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1111741_CREB_Liver_fasted4.bedgraph.gz |
64.2 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
