NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM2228905 Query DataSets for GSM2228905
Status Public on Jul 15, 2016
Title H3K36me3 Chip-Seq KC
Sample type SRA
 
Source name Differentiated Keratinocytes
Organism Homo sapiens
Characteristics passage: 3
tissue: Foreskin
chip-antibody: H3K36me3 (Abcam Cat: ab9050)
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
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.
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)
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
 
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 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.
 
Submission date Jul 08, 2016
Last update date May 15, 2019
Contact name Debayan Datta
Organization name IRB Barcelona
Department Oncology
Lab Stem Cells and Cancer
Street address C. Baldiri Reixac 10
City Barcelona
State/province Barcelona
ZIP/Postal code 08028
Country Spain
 
Platform ID GPL16791
Series (1)
GSE65838 Dnmt3a and Dnmt3b associate with enhancers to regulate human epidermal stem cell homeostasis
Relations
BioSample SAMN05368352
SRA SRX1916927

Supplementary file Size Download File type/resource
GSM2228905_H3K36me3_KC_peaks.bed.gz 1007.6 Kb (ftp)(http) BED
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap