|
| Status |
Public on Nov 24, 2010 |
| Title |
EC_NucPos_WT_rep#1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Cells grown in YS
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: h-, 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-, 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 |
|
