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Sample GSM1262660 Query DataSets for GSM1262660
Status Public on Nov 13, 2013
Title 5C-H3K27me3-ChIP
Sample type genomic
 
Channel 1
Source name H3K27me3 ChIP DNA from HCC specimens X5C
Organism Homo sapiens
Characteristics tissue: HCC specimens and their adjacent tissues
Treatment protocol HCC specimens were transported by dry ice.
Growth protocol Fresh HCC specimens were at -80°C.
Extracted molecule genomic DNA
Extraction protocol Grind the tissue(50-100mg) into a fine powder by Biopulverizer. Add 10ml of culture medium and tissue to the dish. Crosslink by adding 270ul of 37% formaldehyde to a final concentration of 1% to the dish in the hood. Incubate for 10 minutes at room temperature(RT) on orbital shaker. Followed by quenching with 0.125 M glycine for 5 min. The tissue 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 HCC specimens X4C
Organism Homo sapiens
Characteristics tissue: HCC specimens and their adjacent tissues
Treatment protocol HCC specimens were transported by dry ice.
Growth protocol Fresh HCC specimens were at -80°C.
Extracted molecule genomic DNA
Extraction protocol Grind the tissue(50-100mg) into a fine powder by Biopulverizer. Add 10ml of culture medium and tissue to the dish. Crosslink by adding 270ul of 37% formaldehyde to a final concentration of 1% to the dish in the hood. Incubate for 10 minutes at room temperature(RT) on orbital shaker. Followed by quenching with 0.125 M glycine for 5 min. The tissue 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 HCC specimens 5C
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)
GSE52301 Chip-chip from Hepatocellular carcinoma (HCC) specimens with H3K27me3

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

Data table
ID_REF VALUE
CHR10P100019697 -0.0476822861864133
CHR10P100017997 1.15449688911669
CHR10P100019097 -0.0764894007076661
CHR10P100018697 0.135012019317699
CHR10P100017897 0.951923943724144
CHR10P100019797 0.204214299847008
CHR10P100018397 0.70671930450594
CHR10P100017497 0.617121682578255
CHR10P100018897 -0.484362951387216
CHR10P100019497 -0.351457219927086
CHR10P100019897 0.314856816773488
CHR10P100018197 1.16336537619562
CHR10P100020097 -0.49497963685253
CHR10P100019297 -0.0418854391526028
CHR10P100019597 -0.532623759548236
CHR10P100018797 -0.0660493662782572
CHR10P100019197 -0.0160941916972843
CHR10P100017597 0.605259736155994
CHR10P100019997 -0.375526051107271
CHR10P100018497 0.731615204601016

Total number of rows: 386230

Table truncated, full table size 12985 Kbytes.




Supplementary file Size Download File type/resource
GSM1262660_5C.jpg.gz 4.8 Mb (ftp)(http) JPG
GSM1262660_5C_532.pair.gz 7.1 Mb (ftp)(http) PAIR
GSM1262660_5C_635.pair.gz 7.0 Mb (ftp)(http) PAIR
GSM1262660_X5C_635_ratio.gff.gz 7.9 Mb (ftp)(http) GFF
GSM1262660_X5C_635_ratio_peaks.gff.gz 247.6 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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