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Sample GSM1262655 Query DataSets for GSM1262655
Status Public on Nov 13, 2013
Title HepG2 H3K27me3-ChIP
Sample type genomic
 
Channel 1
Source name H3K27me3 ChIP DNA from HepG2 cells
Organism Homo sapiens
Characteristics cell line: HepG2
cell type: Hepatocellular carcinoma
Treatment protocol HepG2 cells were passaged to a new dish and cultured 2 days
Growth protocol HepG2 cell were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Which were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin–streptomycin.
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.
Label Cy5
Label protocol 1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
 
Channel 2
Source name Input DNA from HepG2 cells
Organism Homo sapiens
Characteristics cell line: HepG2
cell type: Hepatocellular carcinoma
Treatment protocol HepG2 cells were passaged to a new dish and cultured 2 days
Growth protocol HepG2 cell were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Which were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin–streptomycin.
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.
Label Cy3
Label protocol 1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
 
 
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)
Scan protocol Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
Description H3K27me3 ChIP in HepG2 cells
Data processing Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction.
 
Submission date Nov 12, 2013
Last update date Nov 13, 2013
Contact name Shubin Gao
E-mail(s) shbgao@xmu.edu.cn
Phone +86-592-218-1535
Organization name Medical College,Xiamen University
Department Basic Medical Sciences
Street address Chengzhi building 110,Xiang'an South Road
City Xiamen
ZIP/Postal code 361102
Country China
 
Platform ID GPL9448
Series (1)
GSE52300 Chip-chip from HepG2 cells with EZH2, SUZ12 and H3K27me3

Data table header descriptions
ID_REF
VALUE scaled, log2 (ChIP/Input) ratio

Data table
ID_REF VALUE
CHR10P100019697 -0.554223497125356
CHR10P100017997 1.61294789939706
CHR10P100019097 -0.444087654512654
CHR10P100018697 0.283802106111284
CHR10P100017897 0.65236417290526
CHR10P100019797 -0.815264160694839
CHR10P100018397 0.364220768198449
CHR10P100017497 -0.708656692589584
CHR10P100018897 1.25907594871737
CHR10P100019497 -0.158879135991083
CHR10P100019897 -0.956037146655468
CHR10P100018197 0.513279729999843
CHR10P100020097 -0.410402817214629
CHR10P100019297 0.59190210064559
CHR10P100019597 -0.422922727843684
CHR10P100018797 0.749434487442787
CHR10P100019197 -0.10305264237105
CHR10P100017597 -0.586819058949543
CHR10P100019997 -0.693224721223969
CHR10P100018497 0.38981181744283

Total number of rows: 386230

Table truncated, full table size 12941 Kbytes.




Supplementary file Size Download File type/resource
GSM1262655_xubin_H3K27.jpg.gz 4.0 Mb (ftp)(http) JPG
GSM1262655_xubin_H3K27_532.pair.gz 7.1 Mb (ftp)(http) PAIR
GSM1262655_xubin_H3K27_635.pair.gz 6.9 Mb (ftp)(http) PAIR
GSM1262655_xubin_H3K27_635_ratio.gff.gz 8.0 Mb (ftp)(http) GFF
GSM1262655_xubin_H3K27_635_ratio_peaks.gff.gz 152.9 Kb (ftp)(http) GFF
Processed data included within Sample table
Processed data provided as supplementary file
Processed data are available on Series record

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