|
| Status |
Public on Dec 12, 2014 |
| Title |
Set1_1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Schizosaccharomyces pombe cells, Set1 wt ChIP
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: wildtype
|
| Treatment protocol |
50 mL cultures were cross-linked with paraformadehyde at 18 deg C for 30 minutes, followed by dimethyl adipimidate crosslinking at room temperature for 45 min.
|
| Growth protocol |
Logarithmically growing cultures were grown 18 hours to mid-exponential phase (A595 ~ 0.5) in standard rich media (YEA) shaking at 250 RPM at 30° C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was extracted from cross-linked cells following bead beater lysis and sonication. Following immunoprecipitation of ChIP samples, ChIP and input DNA samples were incubated overnight at 65 deg C in TE buffer to reverse cross-links and digested with Rnase A and proteinase K. DNA was recovered by phenol/chloroform extraction and ethanol precipitation
|
| Label |
Alexa Fluor 647
|
| Label protocol |
ChIP and Input DNA were amplified by random priming PCR incorporating amino-allyl dUTP, then covalently coupled with Cy5 mono NHS ester in 100 mM sodium bicarbonate for 1 hr at room temperature. Labeled DNA was recovered using Qiagen QIAquick PCR columns.
|
| |
|
| Channel 2 |
| Source name |
Schizosaccharomyces pombe cells, input
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: wildtype
|
| Treatment protocol |
50 mL cultures were cross-linked with paraformadehyde at 18 deg C for 30 minutes, followed by dimethyl adipimidate crosslinking at room temperature for 45 min.
|
| Growth protocol |
Logarithmically growing cultures were grown 18 hours to mid-exponential phase (A595 ~ 0.5) in standard rich media (YEA) shaking at 250 RPM at 30° C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was extracted from cross-linked cells following bead beater lysis and sonication. Following immunoprecipitation of ChIP samples, ChIP and input DNA samples were incubated overnight at 65 deg C in TE buffer to reverse cross-links and digested with Rnase A and proteinase K. DNA was recovered by phenol/chloroform extraction and ethanol precipitation
|
| Label |
Alexa Fluor 555
|
| Label protocol |
ChIP and Input DNA were amplified by random priming PCR incorporating amino-allyl dUTP, then covalently coupled with Cy5 mono NHS ester in 100 mM sodium bicarbonate for 1 hr at room temperature. Labeled DNA was recovered using Qiagen QIAquick PCR columns.
|
| |
|
| |
| Hybridization protocol |
500 ng of AlexaFluor 555 labeled Input DNA was mixed with 500 ng of AlexaFluro 647 labeled ChIP DNA plus 50 ul Agilent 10X control targets and 250 ul of 2X Agilent Hybridization buffer. Combined samples were hybridized to Agilent 4x44k array slides in an Agilent SureHyb chamber at 65° C, 20 RPM for 24 hours. Slides were washed once with 6X SSPE. (Invitrogen)/0.005% N-Lauroylsarcosine (Sigma L7414), once with 0.06X SSPE/0.005% N-Lauroylsarcosine, and once with Agilent Stabilization and Drying solution according to Agilent protocols.
|
| Scan protocol |
Slides were scanned using an Agilent G2565CA Scanner.
|
| Description |
ChIP-chip profiling of Set1 in wildtype cells replicate 1
|
| Data processing |
Scanned slides were processed using Agilent Feature Extraction Software (v.10.5.1.1), extraction protocol GE2_105_Jul10. Red and green channel spot and background intensities from Feature Extraction files were processed using the R/Bioconductor Ringo package (v1.3.0) using the read.maimages function (source=agilent). ChIP vs. input ratios (log2) were determined with the preprocess function using the loess normalization method.
|
| |
|
| Submission date |
Nov 14, 2014 |
| Last update date |
Dec 12, 2014 |
| Contact name |
Hugh P Cam |
| E-mail(s) |
hugh.cam@bc.edu
|
| Phone |
6175526851
|
| Organization name |
Boston College
|
| Department |
Biology
|
| Street address |
Higgins Hall Room 355, 140 Commonwealth Avenue
|
| City |
Chestnut Hill |
| State/province |
MA |
| ZIP/Postal code |
02467 |
| Country |
USA |
| |
|
| Platform ID |
GPL6503 |
| Series (1) |
| GSE63301 |
Heterochromatin assembly and transcriptome repression by Set1 in coordination with a class II histone deacetylase |
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