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| Status |
Public on Jul 07, 2015 |
| Title |
Liver_1h_rep3 |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
NF-kB ChIP DNA from Rattus norvegicus regenerating liver at 1h post PHx, biological replicate 3
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| Organism |
Rattus norvegicus |
| Characteristics |
genotype: Wild-type
gender: Male
strain: Sprague Dawley
age: 8-10 weeks
weight: 275-300 g
liver lobe: Right median, right lateral, and caudate lobe
chip antibody: NF-κB antibody (Cat#ab7970, Rabbit polyclonal NF-κB p65 antibody and negative control IgG antibody from Abcam Inc, Cambridge, MA)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5x10E6 cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for at least 30 min on ice. The crosslinked chromatin was sheared to an average size of 500 bp by ten 30-second pulses using a BiorupterTM sonicator (Diagenode). The lysate was then centrifuged to remove cell debris, and the chromatin was quantified in a UV spectrophotometer. The chromatin (400 µg) was diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and pre-cleared with 25 µl protein A sepharose beads (pre-absorbed with salmon sperm DNA and BSA) for 2 h at 4C. The chromatin supernatant was then incubated overnight with 5 µg of purified antibody at 4C. After incubation with protein A sepharose beads (50 µl) for 2h at 4C, the immune complexes were collected by centrifugation and washed with the following buffers each for 10 min at 4C; RIPA buffer (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS, 1% NP40) LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and 2x TE (20 mM Tris pH8.0, 2 mM EDTA). The protein-DNA complexes were eluted from the beads in 450 µl elution buffer (1% SDS, 100 mM NaHCO3) at 55C for 2 h followed by the addition of proteinaseK to 500 µg/ml and overnight incubaton at 65C. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input (1/100 of the material used for ChIP) by phenol extraction and ethanol precipitation.
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| Label |
Cy5
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| Label protocol |
1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy5 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
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| Channel 2 |
| Source name |
Input DNA from Rattus norvegicus regenerating liver at 1h post PHx, biological replicate 3
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| Organism |
Rattus norvegicus |
| Characteristics |
genotype: Wild-type
gender: Male
strain: Sprague Dawley
age: 8-10 weeks
weight: 275-300 g
liver lobe: Right median, right lateral, and caudate lobe
chip antibody: none, input DNA
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5x10E6 cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for at least 30 min on ice. The crosslinked chromatin was sheared to an average size of 500 bp by ten 30-second pulses using a BiorupterTM sonicator (Diagenode). The lysate was then centrifuged to remove cell debris, and the chromatin was quantified in a UV spectrophotometer. The chromatin (400 µg) was diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and pre-cleared with 25 µl protein A sepharose beads (pre-absorbed with salmon sperm DNA and BSA) for 2 h at 4C. The chromatin supernatant was then incubated overnight with 5 µg of purified antibody at 4C. After incubation with protein A sepharose beads (50 µl) for 2h at 4C, the immune complexes were collected by centrifugation and washed with the following buffers each for 10 min at 4C; RIPA buffer (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS, 1% NP40) LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and 2x TE (20 mM Tris pH8.0, 2 mM EDTA). The protein-DNA complexes were eluted from the beads in 450 µl elution buffer (1% SDS, 100 mM NaHCO3) at 55C for 2 h followed by the addition of proteinaseK to 500 µg/ml and overnight incubaton at 65C. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input (1/100 of the material used for ChIP) by phenol extraction and ethanol precipitation.
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| Label |
Cy3
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| Label protocol |
1 µg Input DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
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| Hybridization protocol |
The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)
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| Scan protocol |
Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
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| Description |
ChIP-chip Regenerating rat liver, 1h, NFkB
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| Data processing |
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction. Array images were used for data extraction as paired files; genomic feature format files were then produced for analysis of scaled log2-ratio data. The intensity ratio of immunopreciptated to total DNA (not taken through immunoprecipitation steps) was calculated at each genomic position to identify regions where increased signal (i.e. DNA fragment enrichment) was observed relative to the control sample. Peak regions identified as statistically significant binding sites were generated from the scaled log2-ratio data. The possible binding regimes (peaks) that correspond to the binding targets were detected if 4 or more probes showed a signal above the cutoff value ranging from 90% to 15% using a 500bp sliding window. Cut-off values were set as a fraction of the hypothetical maximum signal (mean signal plus six standard deviations). An empirical false discovery rate (FDR) was calculated for each peak using a bootstrapping method, which randomized the probe signals 20 times. The calculated FDR is an approximation of the probability of a false positive. Peaks were annotated with candidate target genes with the assumption that the distance between a center of binding peak and the transcription start site (TSS) of the gene is shorter than a threshold cutoff. We defined these “gene regions” as spanning from 5kb upstream of the TSS to 1.5 kb downstream of the end of transcription. The peak files were annotated with Ensembl version 5.0 (Rnor_5.0) transcript genes using a 5000 base pair cutoff distance from the TSS using the Chip Peak Anno Bioconductor package in R. The peaks were then annotated with detailed characteristic genomic features: peak region location, gene annotation of nearest transcript (intron, exon, intragenic, etc.), chromosome, start and end of genes, nearest transcripts and transcript boundaries, distance from TSS, RefSeq IDs, Entrez IDs, TSS, trophoblast-specific element (TSE) and average phastcon scores obtained.
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| Submission date |
Jul 06, 2015 |
| Last update date |
Jul 07, 2015 |
| Contact name |
Daniel J Cook |
| E-mail(s) |
djcook@udel.edu
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| Phone |
2155032969
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| Organization name |
University of Delaware
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| Department |
Chemical and Biomolecular Engineering
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| Lab |
Ogunnaike
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| Street address |
104 Academy St.
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| City |
Newark |
| State/province |
DE |
| ZIP/Postal code |
19716 |
| Country |
USA |
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| Platform ID |
GPL20661 |
| Series (1) |
| GSE70522 |
A Novel, Dynamic Pattern-based Analysis of NF-kappaB Binding During the Priming Phase of Liver Regeneration Reveals Switch-Like Functional Regulation |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1808348_469328_DOUBLEPASS_REGION2_Green.pair.gz |
12.3 Mb |
(ftp)(http) |
PAIR |
| GSM1808348_469328_DOUBLEPASS_REGION2_Red.pair.gz |
12.3 Mb |
(ftp)(http) |
PAIR |
| GSM1808348_469328_DOUBLEPASS_REGION2_Red_ratio_peaks_mapToFeatures_All_Peaks_PHx1_h-3.txt.gz |
298.9 Kb |
(ftp)(http) |
TXT |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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