NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM488327 Query DataSets for GSM488327
Status Public on Nov 24, 2010
Title EC_NucPos_Clr4_rep#1
Sample type genomic
 
Channel 1
Source name Cells grown in YS
Organism Schizosaccharomyces pombe
Characteristics strain: h-, clr4::kanMX, smt0, ade6-M210, leu1-32, ura4-D18
sample type: input
Extracted molecule genomic DNA
Extraction protocol Cells were grown to ~0.75-0.80 OD600 in YS at 30°C and crosslinked for 20 min. at 30°C using 1% formaldehyde followed by a 0.125M glycine quench for 5 min. Seventy-five to eighty OD600 units of cells were resuspended with 0.5mg Zymolase 100-T in 0.5mL of CES buffer. The cells were spheroplasted at 30°C for 1 hr. on a rocker. Spheroplasts were collected by centrifugation at 3000 x g for 3 min. at 4°C then washed with cold 1.2M sorbitol twice. The spheroplasts were then resuspended in a total volume of 0.8mL using pNP-S buffer. The source material for this channel was obtained from 0.2mL of resuspended spheroplasts that was added and mixed with 0.3mL of pNP-S buffer containing 0 units of MNase and then was incubated at at 37°C for 15 minutes. The reaction was then quenched by adding EDTA pH 8.0 to a final concentration of 50mM. The spheroplasts were lysed with 0.1% SDS and centrifuged at 20k x g for 5 minutes. The supernatant containing solublized chromatin was incubated at 65°C overnight with 0.4 mg/ml proteinase K to deproteinize DNA and reverse methylene crosslinks. DNA was recovered by two extractions with equal amounts of equilibrated phenol and chloroform followed by ethanol precipitation and resuspension in water supplemented with 10μg/mL RNase A. After a 30 min. treatment at 37°C, approximately 80μg DNA was then treated lightly with 1.5U of MNase for 15 minutes. After a phenol::chloroform extraction and ethanol precipitation, the DNA was amplified and labeled with a Cy dye using an Invitrogen Bioprime protocol and a high concentration of Klenow exo- and random nonamer primers.
Label Cy3
Label protocol Amplified material containing aminoallyl-dUTP was coupled to the indicated Cy dye according to manufacturer (Amersham Biosciences) instructions.
 
Channel 2
Source name Cells grown in YS
Organism Schizosaccharomyces pombe
Characteristics strain: h-, clr4::kanMX, smt0, ade6-M210, leu1-32, ura4-D18
sample type: MNase treated genomic DNA
Extracted molecule genomic DNA
Extraction protocol Cells were grown to ~0.75-0.80 OD600 in YS at 30°C and crosslinked for 20 min. at 30°C using 1% formaldehyde followed by a 0.125M glycine quench for 5 min. Seventy-five to eighty OD600 units of cells were resuspended with 0.5mg Zymolase 100-T in 0.5mL of CES buffer. The cells were spheroplasted at 30°C for 1 hr. on a rocker. Spheroplasts were collected by centrifugation at 3000 x g for 3 min. at 4°C then washed with cold 1.2M sorbitol twice. The spheroplasts were then resuspended in a total volume of 0.8mL using pNP-S buffer. The source material for this channel was obtained from 0.2mL of resuspended spheroplasts that was added and mixed with 0.3mL of pNP-S buffer containing 150 units of MNase and then was incubated at at 37°C for 15 minutes. The reaction was then quenched by adding EDTA pH 8.0 to a final concentration of 50mM. The spheroplasts were lysed with 0.1% SDS and centrifuged at 20k x g for 5 minutes. The supernatant containing solublized chromatin was incubated at 65°C overnight with 0.4 mg/ml proteinase K to deproteinize DNA and reverse methylene crosslinks. DNA was recovered by two extractions with equal amounts of equilibrated phenol and chloroform followed by ethanol precipitation and resuspension in water supplemented with 10μg/mL RNase A. After a 30 min. treatment at 37°C, the DNA was amplified and labeled with a Cy dye using an Invitrogen Bioprime protocol and a high concentration of Klenow exo- and random nonamer primers.
Label Cy5
Label protocol Amplified material containing aminoallyl-dUTP was coupled to the indicated Cy dye according to manufacturer (Amersham Biosciences) instructions.
 
 
Hybridization protocol Hybridizations and washes were conducted in accordance to the Agilent Yeast ChIP-on-chip Analysis protocol. 1450 ng of material was used for each channel.
Scan protocol Arrays were scanned at a resolution of 5 pixels/um on an Axon 4000B Scanner using GenePix Pro 5.0 software
Description No further descriptions
Data processing Data were corrected for background by subtracting the median background value. Data were then normalized using a Loess algorithm.
 
Submission date Dec 21, 2009
Last update date Nov 24, 2010
Contact name Hiten Madhani
E-mail(s) hitenmadhani@gmail.com
URL http://madhanilab.ucsf.edu
Organization name University of California, San Francisco
Department Biochemistry and Biophysics
Street address 600 16th St.
City San Francisco
State/province CA
ZIP/Postal code 94143-2200
Country USA
 
Platform ID GPL9819
Series (1)
GSE19596 Silencing factors induce the elimination of nucleosome-free regions in S. pombe heterochromatin

Data table header descriptions
ID_REF
VALUE log2 mononucleosome:genomic ratio that has been smoothed with a centering average using a window of 19 probes

Data table
ID_REF VALUE
12 0.322648521
13 0.324490632
14 0.279286506
15 -0.566707105
16 0.063426015
17 -0.401169842
18 0.637939421
19 0.411903368
20 0.267927204
21 0.285147211
22 -0.315796084
23 -0.377176047
24 0.270819953
25 0.122863989
26 0.055447918
27 0.335545119
28 -0.240841
29 -0.686432263
30 0.296300874
31 -0.078543722

Total number of rows: 42683

Table truncated, full table size 755 Kbytes.




Supplementary file Size Download File type/resource
GSM488327_EC_NucPos_Clr4_rep_1.gpr.gz 4.4 Mb (ftp)(http) GPR
Processed data included within Sample table

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