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Status |
Public on Jun 09, 2016 |
Title |
BrdU IP in cdc25-22 synchronized delta clr4 cells 120 min |
Sample type |
genomic |
|
|
Channel 1 |
Source name |
cdc25-22 delta clr4_synchronized_IP DNA
|
Organism |
Schizosaccharomyces pombe |
Characteristics |
genotype/variation: cdc25-22 ∆clr4 cells molecule subtype: BrdU immunoprecipitated DNA
|
Treatment protocol |
Cells were collected at time intervals corresponding to maximum septation index and fixed with 0.1% sodium azide.
|
Growth protocol |
Cdc25-22 cells carrying thymidine kinase expression module Pnmt1-TK and nucleoside transport module Padh1-hENT were grown in minimal media at 26˚C and arrested at the G2/M block by raising temperature to 37˚C for 4h and 15 min. Cells were released from block by reducing temperature to 26˚C, and cultures were supplemented at this time by 10mM hydroxyurea and 200μΜ 5-bromo-2'-deoxyuridine.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
The DNA was phenol-extracted from fixed cells by bead-beating with glass beads, sheared by sonication to 500-1000bp fragments and immunoprecipitated with anti-BrdU antibody (BD Pharmigen) immobilized to Dynal anti-mouse magnetic beads (Invitrogen).
|
Label |
Cy5
|
Label protocol |
Immunoprecipitated and whole-cell extract DNA was amplified by random-primed PCR and conjugated with Cy5 (IP DNA) or Cy3 (whole-cell extract DNA).
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|
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Channel 2 |
Source name |
cdc25-22 ∆clr4_synchronized_whole-cell extract
|
Organism |
Schizosaccharomyces pombe |
Characteristics |
genotype/variation: cdc25-22 delta clr4 cells molecule subtype: Whole-cell extract DNA
|
Treatment protocol |
Cells were collected at time intervals corresponding to maximum septation index and fixed with 0.1% sodium azide.
|
Growth protocol |
Cdc25-22 cells carrying thymidine kinase expression module Pnmt1-TK and nucleoside transport module Padh1-hENT were grown in minimal media at 26˚C and arrested at the G2/M block by raising temperature to 37˚C for 4h and 15 min. Cells were released from block by reducing temperature to 26˚C, and cultures were supplemented at this time by 10mM hydroxyurea and 200μΜ 5-bromo-2'-deoxyuridine.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
The DNA was phenol-extracted from fixed cells by bead-beating with glass beads, sheared by sonication to 500-1000bp fragments and immunoprecipitated with anti-BrdU antibody (BD Pharmigen) immobilized to Dynal anti-mouse magnetic beads (Invitrogen).
|
Label |
Cy3
|
Label protocol |
Immunoprecipitated and whole-cell extract DNA was amplified by random-primed PCR and conjugated with Cy5 (IP DNA) or Cy3 (whole-cell extract DNA).
|
|
|
|
Hybridization protocol |
Equal amounts of Cy5-labeled ChIP DNA and Cy3-labeled whole-cell extract DNA were mixed and combined with human Cot1 DNA, Agilent Blocking Agent and Agilent Hybridization buffer, and hybridized to high-density microarrays in Agilent SureHyb hybridization chamber for 24 hours at 65˚C, 10 rpm. After hybridization, slides were washed according to Agilent protocol.
|
Scan protocol |
Scanned on an Agilent G2505B scanner.
|
Description |
BrdU IP ∆clr4 120min V3 series1
|
Data processing |
Data were extracted using Agilent Feature Extraction Software (CHIP_1105_Oct12 ). Signal was normalized by combined rank consistency filtering with LOWES intensity normalization. Enrichment values were calculated as a ratio of Cy5 processed signal/ Cy3 processed signal.
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Submission date |
Mar 02, 2016 |
Last update date |
Jun 10, 2016 |
Contact name |
Shiv Grewal |
Phone |
2407607553
|
Organization name |
NCI
|
Department |
LBMB
|
Lab |
Shiv Grewal
|
Street address |
NCI bldg 37 Rm 6068 9000 Rockville Pike
|
City |
Bethesda |
State/province |
MD |
ZIP/Postal code |
20892 |
Country |
USA |
|
|
Platform ID |
GPL6503 |
Series (2) |
GSE78819 |
Taz1-Shelterin promotes facultative heterochromatin assembly at chromosome-internal sites containing late replication origins [BrdU] |
GSE78823 |
Taz1-Shelterin promotes facultative heterochromatin assembly at chromosome-internal sites containing late replication origins |
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