|
| Status |
Public on Jan 30, 2017 |
| Title |
Crypts_Diabetes_16weeks_rep2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Promoter methylation DNA from intestinal crypts in 16 weeks old diabetic db/db mice
|
| Organism |
Mus musculus |
| Characteristics |
background strain: BKS.Cg-Dock7m +/+ Leprdb/JNju
tissue: intestinal crypt
antibody: Diagenode, C15200081, GF-004
age: 16 weeks
genotype: db/db
phenotype: obese
blood glucose value: ≥ 16.7 mmol/l
|
| Treatment protocol |
Gene Lepr was mutated in single allele.
|
| Growth protocol |
Mice were maintained under a 12 h light/12 h dark cycle in a specific pathogen-free animal facility.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (gDNA) was extracted from 6 samples using a DNeasy Blood & Tissue Kit (Qiagen, Fremont, CA). The purified gDNA was then quantified and quality assessed by nanodrop ND-1000. Genomic DNA of each sample was sonicated to ~200 – 1000 bp with a Bioruptor sonicator (Diagenode) on “Low” mode for 10 cycles of 30 seconds “ON” & 30 seconds “OFF”. The gDNA and each sheared DNA were aragrose analyzed.
|
| Label |
Cy5
|
| Label protocol |
For DNA labelling, the NimbleGen Dual-Color DNA Labeling Kit was used according to the manufacturer’s guideline detailed in the NimbleGen MeDIP-chip protocol (NimbleGen Systems, Inc., Madison, WI, USA). 1 μg DNA of each sample was incubated for 10 min at 98°C with 1 OD of Cy5-9mer primer (IP sample) or Cy3-9mer primer (Input sample). Then, 100 pmol of deoxynucleoside triphosphates and 100U of the Klenow fragment (New England Biolabs, USA) were added and the mix incubated at 37°C for 2 hours. The reaction was stopped by adding 0.1 volume of 0.5 M EDTA, and the labeled DNA was purified by isopropanol / ethanol precipitation.
|
| |
|
| Channel 2 |
| Source name |
Input DNA from D2
|
| Organism |
Mus musculus |
| Characteristics |
background strain: BKS.Cg-Dock7m +/+ Leprdb/JNju
tissue: intestinal crypt
age: 16 weeks
genotype: db/db
phenotype: obese
blood glucose value: ≥ 16.7 mmol/l
|
| Treatment protocol |
Gene Lepr was mutated in single allele.
|
| Growth protocol |
Mice were maintained under a 12 h light/12 h dark cycle in a specific pathogen-free animal facility.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (gDNA) was extracted from 6 samples using a DNeasy Blood & Tissue Kit (Qiagen, Fremont, CA). The purified gDNA was then quantified and quality assessed by nanodrop ND-1000. Genomic DNA of each sample was sonicated to ~200 – 1000 bp with a Bioruptor sonicator (Diagenode) on “Low” mode for 10 cycles of 30 seconds “ON” & 30 seconds “OFF”. The gDNA and each sheared DNA were aragrose analyzed.
|
| Label |
Cy3
|
| Label protocol |
For DNA labelling, the NimbleGen Dual-Color DNA Labeling Kit was used according to the manufacturer’s guideline detailed in the NimbleGen MeDIP-chip protocol (NimbleGen Systems, Inc., Madison, WI, USA). 1 μg DNA of each sample was incubated for 10 min at 98°C with 1 OD of Cy5-9mer primer (IP sample) or Cy3-9mer primer (Input sample). Then, 100 pmol of deoxynucleoside triphosphates and 100U of the Klenow fragment (New England Biolabs, USA) were added and the mix incubated at 37°C for 2 hours. The reaction was stopped by adding 0.1 volume of 0.5 M EDTA, and the labeled DNA was purified by isopropanol / ethanol precipitation.
|
| |
|
| |
| Hybridization protocol |
Microarrays were hybridized at 42°C during 16 to 20h with Cy3/5 labelled DNA in NimbleGen hybridization buffer/ hybridization component A in a hybridization chamber (Hybridization System - NimbleGen Systems, Inc., Madison, WI, USA). Following hybridization, washing was performed using the NimbleGen Wash Buffer kit (NimbleGen Systems, Inc., Madison, WI, USA).
|
| Scan protocol |
From the normalized log2-ratio data, a sliding-window peak-finding algorithm provided by NimbleScan v2.5 (Roche-NimbleGen) was applied to find the enriched peaks with specified parameters (sliding window width: 1,500bp; mini probes per peak: 2; P-value minimum cutoff: 2; maximum spacing between nearby probes within peak: 500bp).
|
| Description |
Sample name: D2
It is the second of three db/db biological replicates used in this experimen.
meDIP-chip
|
| Data processing |
We perform Median-centering, quantile normalization and linear smoothing in this analysis process by Bioconductor packages Ringo, limma, and MEDME. After normalization, a normalized log2-ratio data (*_ratio.gff file) was created for each sample, it can be used to further peak-finding analysis.
|
| |
|
| Submission date |
Sep 17, 2016 |
| Last update date |
Jan 30, 2017 |
| Contact name |
Can-Ze Huang |
| E-mail(s) |
sunshinehcz@163.com
|
| Organization name |
Sun Yat-Sen Memorial Hospital
|
| Street address |
107 Yan-Jiang Xi-Road
|
| City |
Guang-Zhou |
| ZIP/Postal code |
510120 |
| Country |
China |
| |
|
| Platform ID |
GPL21852 |
| Series (1) |
| GSE87044 |
Sox9 transcriptionally regulates Wnt signaling in intestinal epithelium stem cells under hypomethylated crypts in diabetic state |
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