|
| Status |
Public on Mar 21, 2017 |
| Title |
BrdU IP in cdc25-22synchronized fft3∆ cells 50min (HU) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
cdc25 synchronized cells_BrdU immunoprecipitated DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: fft3_delta
|
| 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 25˚C in water incubator with rotation of 140 rpm, and cultures were supplemented at this time by 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 Pharmingen, 555627) 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).
|
| |
|
| Channel 2 |
| Source name |
cdc25 synchronized cells_whole-cell extract DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: fft3_delta
|
| 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 25˚C in water incubator with rotation of 140 rpm, and cultures were supplemented at this time by 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 Pharmingen, 555627) 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.
|
| Data processing |
Data were extracted using Agilent Feature Extraction Software (ChIP_1200_Jun14 ). Signal was normalized by combined rank consistency filtering with LOWES intensity normalization.
|
| |
|
| Submission date |
Jan 06, 2017 |
| Last update date |
Mar 21, 2017 |
| 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) |
| GSE88904 |
Snf2 Family Protein Fft3 Suppresses Nucleosome Turnover to Promote Epigenetic Inheritance of Heterochromatin and Proper Replication of the Genome |
| GSE93273 |
Snf2 Family Protein Fft3 Suppresses Nucleosome Turnover to Promote Epigenetic Inheritance of Heterochromatin and Proper Replication of the Genome [HU + BrdU IP] |
|
