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Sample GSM1260933 Query DataSets for GSM1260933
Status Public on Nov 08, 2013
Title X1C-H3K4me3-ChIP
Sample type genomic
 
Channel 1
Source name H3K4me3 ChIP DNA from HCC specimens X1C
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 X1C
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 H3K4me3 ChIP in HCC specimens X1C
Data processing Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction.
 
Submission date Nov 07, 2013
Last update date Nov 08, 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)
GSE52190 Chip-chip from Hepatocellular carcinoma (HCC) specimens with H3K4me3

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

Data table
ID_REF VALUE
CHR10P100019697 1.16748849183116
CHR10P100017997 -0.768650347853858
CHR10P100019097 -0.0264433883481323
CHR10P100018697 0.601460919307863
CHR10P100017897 -0.913840258695148
CHR10P100019797 0.943791014805838
CHR10P100018397 0.586460904749722
CHR10P100017497 2.10241133739212
CHR10P100018897 0.250149543060643
CHR10P100019497 0.0184717331994888
CHR10P100019897 0.779962290527713
CHR10P100018197 0.166421861603862
CHR10P100020097 1.26921614927139
CHR10P100019297 0.115565421714176
CHR10P100019597 0.417789893010337
CHR10P100018797 0.356786218269023
CHR10P100019197 0.404779125684942
CHR10P100017597 1.12792265821741
CHR10P100019997 0.805957463818879
CHR10P100018497 0.96664350244701

Total number of rows: 386230

Table truncated, full table size 12929 Kbytes.




Supplementary file Size Download File type/resource
GSM1260933_X1C.jpg.gz 4.5 Mb (ftp)(http) JPG
GSM1260933_X1C_532.pair.gz 7.0 Mb (ftp)(http) PAIR
GSM1260933_X1C_635.pair.gz 7.0 Mb (ftp)(http) PAIR
GSM1260933_X1C_635_ratio.gff.gz 7.9 Mb (ftp)(http) GFF
GSM1260933_X1C_635_ratio_peaks.gff.gz 128.2 Kb (ftp)(http) GFF
Processed data included within Sample table
Processed data are available on Series record

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