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| Status |
Public on Jul 15, 2016 |
| Title |
Dnmt3A Chip-Seq KC |
| Sample type |
SRA |
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| Source name |
Differentiated Keratinocytes
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| Organism |
Homo sapiens |
| Characteristics |
passage: 2
tissue: Foreskin
chip-antibody: Dnmt3a (SantaCruz H-295
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| Treatment protocol |
Epidermal Stem Cells growing in KSM media were infected with precipitated lentiviral particles produced by 293T cells transfected with the mentioned plasmids. Cells were collected for ChIP assay after 5 days of puromycin selection
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| Growth protocol |
Human Epidermal Stem Cells were isolated from neonatal foreskin samples and cultured with a feeder layer of fibroblasts (J2-3T3) as described previously (Gandarillas and Watt, 1997). Undifferentiated epSC were grown in Keratinocyte Serum-Free Medium with supplements (KSFM; GIBCO). For calcium-induced differentiation, after reaching 70% confluence KSFM was exchanged for EMEM (Lonza) supplemented with 8% chelated FBS, EGF (10 ng/ml), 1% penicillin/streptomycin, and 1.2mM CaCl2. After 48 hr differentiated keratinocytes were collected.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
EpiSC and differentiated keratinocytes were trypsinized and crosslinked in 1% formaldehyde (FA) for 10 min at room temperature (RT). For ChIPs from EBs, crosslinking was performed for 30 min at RT in 4% FA. Crosslinking was quenched with 0.125 M glycine. Pelleted cells were lysed in 1 ml ChIP buffer and sonicated for 15 min in a Bioruptor (Diagenode). Soluble material was quantified by Bradford assays. To immunoprecipitate transcription factors, 2000 mg of protein and 100 mg of immunoprecipitated histone/histone modifications were used. Antibodies (10ug for DnmtA/3B and 3ug for histone modifications) were incubated overnight with the chromatin. Immunocomplexes were recovered with 30 ul of protein A bead slurry (Healthcare. Cat # 17-5280-01). Immunoprecipitated material was washed three times with low salt buffer and one time with high salt buffer. DNA complexes were decrosslinked at 65_C overnight, and DNA was then eluted in 50 ul of water using the PCR purification kit (QIAGEN)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
The ChIP-seq datasets were aligned to the human genome build hg19 using Bowtie version 1.01. The parameters used were -k 1, -m 1, -n 2.
Peaks of Dnmt3A and Dnmt3B were called using MACS 1.4.1 using the parameters -p 1e-5, -w, -S and -g hs. For Methylation and HydroxyMethylation peaks, the p-value cutoff was set to 1e-3.
For Histone marks, MACS2 was used with parameters -broad, -q 0.01 -g hs.
Genome_build: hg19
Supplementary_files_format_and_content: The BED files were obtained from the output of MACS/MACS2.
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| Submission date |
Feb 11, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Debayan Datta |
| Organization name |
IRB Barcelona
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| Department |
Oncology
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| Lab |
Stem Cells and Cancer
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| Street address |
C. Baldiri Reixac 10
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| City |
Barcelona |
| State/province |
Barcelona |
| ZIP/Postal code |
08028 |
| Country |
Spain |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE65838 |
Dnmt3a and Dnmt3b associate with enhancers to regulate human epidermal stem cell homeostasis |
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| Relations |
| BioSample |
SAMN03339899 |
| SRA |
SRX873813 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1607392_DNMT3A_KC_peaks.bed.gz |
59.4 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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