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| Status |
Public on Feb 28, 2018 |
| Title |
FT H3ac (non-exposed) |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
H3ac ChIP DNA from non-exposed Pool
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| Organism |
Homo sapiens |
| Characteristics |
gender: Female
tissue: Skin
region: Buttock
chip antibody: H3ac
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| Treatment protocol |
The epidermis and the dermis are separated by incubating the skin fragments overnight at 4 °C in 0.2% dispase solution, then peeling epidermal sheet with forceps.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Epidermis samples were pulverized and chemically crosslinked by 1% formaldehyde. Cells were rinsed twice with PBS and lysed using lysis buffer (0.1% SDS, 0.5% Triton X-100, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl, protease inhibitor, Roche). Lysed cells were sonicated using a Diagenode Bioruptor UCD-200 at high power in ice water. Sonicated fragments ranged in size from 200 to 1000 bp. After sonication, samples were centrifuged at 13,000 ×g for 10 min. The supernatant was pre-absorbed by 50 μl protein A/G beads (Santa Cruz) and incubated with magnetic beads conjugated antibodies (H3 Acetylation Antibody) overnight at 4 °C. The beads were then washed four times by lysis buffer, two times by LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]), and three times by TE buffer. The bound immunocomplex was eluted by adding 300 μl fresh elution buffer (1% SDS, 0.1 M NaHCO3). Twenty microliters of 5M NaCl was then added to the eluted product and incubated at 65 °C overnight to reverse the crosslinking. Immunoprecipitated genomic DNA was then purified using a QIAGEN Purification Kit and dissolved in EB buffer for the following qPCR QC assay or ChIP-chip analysis.
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| Label |
Cy5
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| Label protocol |
The Immunoaffinity-enriched DNA was amplified using a WGA kit from Sigma-Aldrich (GenomePlex® Complete Whole Genome Amplification (WGA2) kit). The amplified DNA samples were then purified with QIAquick PCR purification kit (Qiagen). The purified DNA was quantified using a nanodrop ND-1000. For DNA labelling, the NimbleGen Dual-Color DNA Labeling Kit was used according to the manufacturer’s guideline detailed in the NimbleGen ChIP-on-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.
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| Channel 2 |
| Source name |
Input DNA from non-exposed Pool
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| Organism |
Homo sapiens |
| Characteristics |
gender: Female
tissue: Skin
region: Buttock
chip antibody: none, input DNA
|
| Treatment protocol |
The epidermis and the dermis are separated by incubating the skin fragments overnight at 4 °C in 0.2% dispase solution, then peeling epidermal sheet with forceps.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Epidermis samples were pulverized and chemically crosslinked by 1% formaldehyde. Cells were rinsed twice with PBS and lysed using lysis buffer (0.1% SDS, 0.5% Triton X-100, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl, protease inhibitor, Roche). Lysed cells were sonicated using a Diagenode Bioruptor UCD-200 at high power in ice water. Sonicated fragments ranged in size from 200 to 1000 bp. After sonication, samples were centrifuged at 13,000 ×g for 10 min. The supernatant was pre-absorbed by 50 μl protein A/G beads (Santa Cruz) and incubated with magnetic beads conjugated antibodies (H3 Acetylation Antibody) overnight at 4 °C. The beads were then washed four times by lysis buffer, two times by LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]), and three times by TE buffer. The bound immunocomplex was eluted by adding 300 μl fresh elution buffer (1% SDS, 0.1 M NaHCO3). Twenty microliters of 5M NaCl was then added to the eluted product and incubated at 65 °C overnight to reverse the crosslinking. Immunoprecipitated genomic DNA was then purified using a QIAGEN Purification Kit and dissolved in EB buffer for the following qPCR QC assay or ChIP-chip analysis.
|
| Label |
Cy3
|
| Label protocol |
The Immunoaffinity-enriched DNA was amplified using a WGA kit from Sigma-Aldrich (GenomePlex® Complete Whole Genome Amplification (WGA2) kit). The amplified DNA samples were then purified with QIAquick PCR purification kit (Qiagen). The purified DNA was quantified using a nanodrop ND-1000. For DNA labelling, the NimbleGen Dual-Color DNA Labeling Kit was used according to the manufacturer’s guideline detailed in the NimbleGen ChIP-on-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.
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| Hybridization protocol |
Microarrays were hybridized at 42°C during 16 to 20h with 4μg of 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).
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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 FT epidermal sheet H3ac
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| Data processing |
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction. Processed data files include scaled, log2 (ChIP/Input) ratio.
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| Submission date |
Oct 10, 2017 |
| Last update date |
Feb 28, 2018 |
| Contact name |
Shu Ding |
| Organization name |
The Third Xiangya Hospital
|
| Street address |
Tongzipo street 138#
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| City |
changsha |
| State/province |
hunan |
| ZIP/Postal code |
410000 |
| Country |
China |
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| Platform ID |
GPL15448 |
| Series (1) |
| GSE104764 |
Chip-chip from human epidermis with H3ac |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM2807333_FT_532.pair.gz |
11.9 Mb |
(ftp)(http) |
PAIR |
| GSM2807333_FT_635.pair.gz |
11.8 Mb |
(ftp)(http) |
PAIR |
| GSM2807333_FT_635_ratio.gff.gz |
15.3 Mb |
(ftp)(http) |
GFF |
| GSM2807333_FT_635_ratio_peaks.gff.gz |
228.5 Kb |
(ftp)(http) |
GFF |
| Processed data provided as supplementary file |
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