NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM310842 Query DataSets for GSM310842
Status Public on Sep 11, 2008
Title ChIP_H3K9me2_native_rep2
Sample type genomic
 
Channel 1
Source name ChIP native H3K9me2
Organism Arabidopsis thaliana
Characteristics Col-0, 3 week old, aerial part of the plant
Treatment protocol Plants were frozen in the liquid nitrogen
Growth protocol Plants were grown under continuous light for three weeks.
Extracted molecule genomic DNA
Extraction protocol Native chromatin immunoprecipitation: Plant tissue was resuspended
in 10 ml of HBM buffer (25 mM Tris-Cl pH7.6, 440 mM sucrose, 10 mM MgCl2 and 0.1% Triton-X, 10 mM
β-mercaptoethanol, 2mM spermine, 1 mM PMSF, 1 ug/ml pepstatin and EDTA-free protease inhibitor
cocktail (Roche), homogenized and filtered through Miracloth (Calbiochem). After spinning at 3000 rpm
for 5 min (SS-34, Sorvall), the pellet was resuspended in 5 ml of NIB buffer (20 mM Tris-Cl pH7.6, 250
mM sucrose, 5 mM MgCl2, 5 mM KCl, 0.1% Triton-X, 10 mM β-mercaptoethanol), applied to a 15/50%
Percoll (GE Healthcare) gradient in NIB and spun 2000 rpm for 20 min (SS-34, Sorvall). Isolated nuclei
were washed two times in NIB buffer and flash frozen in liquid nitrogen in HBC buffer (25 mM Tris-Cl
pH7.6, 25 mM Tris-Cl pH7.6, 440 mM sucrose, 10 mM MgCl2 and 0.1% Triton-X, 10 mM β-
mercaptoethanol, 20% glycerol). Nuclei from 1/5 of each preparation were treated with four ul of RNAse
A, 10 ug/ul, (Qiagen) and used for Micrococcal Nuclease (Takara) digestion for 6 minutes (final
concentration 0.2 U/ul) in digestion buffer (16 mM Tris-Cl, pH 7.6, 50 mM NaCl, 2.5 mM CaCl2, 0.01 mM
PMSF and EDTA-free protease inhibitor cocktail (Roche) and stopped with 10 mM EDTA.
Mononucleosomes were released by treating nuclei with 0.1% Triton-X for 1-2 hours in the cold, and then
pelleting the debris by centrifuging at 3500 rpm for 3 min (Eppendorf, 5415R). 500 ul of
supernatant was applied to 50 ul of Dynabeads Protein A (Invitrogen) that was preincubated with 2.5 ug
of the appropriate antibody in buffer (20 mM Tris-Cl, pH 7.6, 50 mM NaCl, 5 mM EDTA and 0.1% Triton) and
incubated overnight. Beads were washed (10 min incubation in the cold) with 500 ul of the
following buffers: 50 mM Tris-Cl pH 7.6, 10 mM EDTA, 0.1 mM PMSF, protease inhibitor cocktail (Roche)
with changing concentration of NaCl – 50 mM, 100 mM and 150 mM. Final wash was done in TE buffer,
without incubation and immunocomplexes were eluted with 500 ul of 0.1% SDS and 0.1 M NaHCO3 at
65 C for 10 min. DNA was then purified using conventional phenol-chlorophorm extraction and
ethanol/salt precipitation. DNA amplification was performed using the GenomePlex® Whole Genome
Amplification Kit (Sigma).
Mouse monoclonal antibody anti-H3K9me2 (Abcam #ab1220; http://www.abcam.com) for immunoprecipitation.

Label Cy5
Label protocol According to standard NimbleGen protocols (www.nimblegen.com)
 
Channel 2
Source name ChIP native H3
Organism Arabidopsis thaliana
Characteristics Col-0, 3 week old, aerial part of the plant
Treatment protocol Plants were frozen in the liquid nitrogen.
Growth protocol Plants were grown under continuous light for three weeks.
Extracted molecule genomic DNA
Extraction protocol Native chromatin immunoprecipitation: Plant tissue was resuspended
in 10 ml of HBM buffer (25 mM Tris-Cl pH7.6, 440 mM sucrose, 10 mM MgCl2 and 0.1% Triton-X, 10 mM
β-mercaptoethanol, 2mM spermine, 1 mM PMSF, 1 ug/ml pepstatin and EDTA-free protease inhibitor
cocktail (Roche), homogenized and filtered through Miracloth (Calbiochem). After spinning at 3000 rpm
for 5 min (SS-34, Sorvall), the pellet was resuspended in 5 ml of NIB buffer (20 mM Tris-Cl pH7.6, 250
mM sucrose, 5 mM MgCl2, 5 mM KCl, 0.1% Triton-X, 10 mM β-mercaptoethanol), applied to a 15/50%
Percoll (GE Healthcare) gradient in NIB and spun 2000 rpm for 20 min (SS-34, Sorvall). Isolated nuclei
were washed two times in NIB buffer and flash frozen in liquid nitrogen in HBC buffer (25 mM Tris-Cl
pH7.6, 25 mM Tris-Cl pH7.6, 440 mM sucrose, 10 mM MgCl2 and 0.1% Triton-X, 10 mM β-
mercaptoethanol, 20% glycerol). Nuclei from 1/5 of each preparation were treated with four ul of RNAse
A, 10 ug/ul, (Qiagen) and used for Micrococcal Nuclease (Takara) digestion for 6 minutes (final
concentration 0.2 U/ul) in digestion buffer (16 mM Tris-Cl, pH 7.6, 50 mM NaCl, 2.5 mM CaCl2, 0.01 mM
PMSF and EDTA-free protease inhibitor cocktail (Roche) and stopped with 10 mM EDTA.
Mononucleosomes were released by treating nuclei with 0.1% Triton-X for 1-2 hours in the cold, and then
pelleting the debris by centrifuging at 3500 rpm for 3 min (Eppendorf, 5415R). 500 ul of
supernatant was applied to 50 ul of Dynabeads Protein A (Invitrogen) that was preincubated with 2.5 ug
of the appropriate antibody in buffer (20 mM Tris-Cl, pH 7.6, 50 mM NaCl, 5 mM EDTA and 0.1% Triton) and
incubated overnight. Beads were washed (10 min incubation in the cold) with 500 ul of the
following buffers: 50 mM Tris-Cl pH 7.6, 10 mM EDTA, 0.1 mM PMSF, protease inhibitor cocktail (Roche)
with changing concentration of NaCl – 50 mM, 100 mM and 150 mM. Final wash was done in TE buffer,
without incubation and immunocomplexes were eluted with 500 ul of 0.1% SDS and 0.1 M NaHCO3 at
65 C for 10 min. DNA was then purified using conventional phenol-chlorophorm extraction and
ethanol/salt precipitation. DNA amplification was performed using the GenomePlex® Whole Genome
Amplification Kit (Sigma).
Rabbit polyclonal antibody anti-H3 (Abcam #ab1791; http://www.abcam.com) was used for immunoprecipitation.
Label Cy3
Label protocol According to standard NimbleGen protocols (www.nimblegen.com)
 
 
Hybridization protocol According to standard NimbleGen protocols (www.nimblegen.com)
Scan protocol According to standard NimbleGen protocols (www.nimblegen.com)
Description no additional information
Data processing Each probe was assigned a log of the ratio between H3K9me2 (Cy5) and H3 (Cy3) signal. Z-scores were calculated for each probe by subtracting the mean log ratio from individual log ratios and dividing by the standard deviation for the entire array.
 
Submission date Aug 07, 2008
Last update date Sep 11, 2008
Contact name Steve E Jacobsen
E-mail(s) yaniki@ucla.edu
Phone (310) 825-0182
URL http://www.mcdb.ucla.edu/Research/Jacobsen/index.html
Organization name University of California, Los Angeles
Department Molecular Cell and Developmental Biology
Lab Jacobsen lab
Street address 621 Charles E. Young Dr. South
City Los Angeles
State/province CA
ZIP/Postal code 90095-1606
Country USA
 
Platform ID GPL7143
Series (1)
GSE12383 Genome wide profiling of H3K9me2 in Arabidopsis thaliana

Data table header descriptions
ID_REF
VALUE log e (Cy5/Cy3)ratio-mean)/standard deviation

Data table
ID_REF VALUE
CHR1V01212004FS000000001 2.588974
CHR1V01212004FS000000061 0.478504
CHR1V01212004FS000000121 1.595237
CHR1V01212004FS000000181 0.060488
CHR1V01212004FS000000241 0.289924
CHR1V01212004FS000000301 -0.060147
CHR1V01212004FS000000361 -0.043945
CHR1V01212004FS000000421 -0.124623
CHR1V01212004FS000000481 -0.267456
CHR1V01212004FS000000541 0.087149
CHR1V01212004FS000000601 0.094086
CHR1V01212004FS000000661 0.016322
CHR1V01212004FS000000721 0.121187
CHR1V01212004FS000000781 0.042395
CHR1V01212004FS000000841 -0.075372
CHR1V01212004FS000000901 -0.035875
CHR1V01212004FS000000961 0.481855
CHR1V01212004FS000001021 0.134015
CHR1V01212004FS000001081 0.270919
CHR1V01212004FS000001141 0.511071

Total number of rows: 2001685

Table truncated, full table size 67685 Kbytes.




Supplementary file Size Download File type/resource
GSM310842_Cy5_Cy3.txt.gz 18.7 Mb (ftp)(http) TXT
Processed data included within Sample table

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