chromatin immunoprecipitation and the chip on chip technique. fredrik fagerström billai b e a- core...
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Chromatin Immunoprecipitation and the
Chip on Chip technique.
Fredrik Fagerström BillaiB E A- Core Facility forB E A- Core Facility for
BBioinformatics and ioinformatics and EExpression xpression AAnalysis.nalysis.Department of Biosciences & Nutrition Department of Biosciences & Nutrition
Karolinska InstitutetKarolinska Institutet
Agenda
-General introduction to Chromatin and Transcriptional and Epigenetic control.
-Chromatin Imunnoprecipitation, tiling arrays and the ”Chip on Chip” technique
Fredrik Fagerström Billai , BEA
Fredrik Fagerström Billai , BEA
A = 30nm fiber of an interphase chromosomeB = Nucleosomes along a strand of DNA
Images of Chromatin
Fredrik Fagerström Billai , BEA
Histones are modified at Specific Amino Acids
Histone Code hypothesis - Modifications of the Histone tails act as marks that can be read by other proteins to control the expression or replication of chromosomal regions. The coding in the histones may
be heritable.
Fredrik Fagerström Billai , BEA
Specific modifications are associated with specific functions.
Transcriptional gene silencing
Chromatin compaction
Genome stability
X chromosome inactivation (females)
Fredrik Fagerström Billai , BEA
Methylation of DNA
Fredrik Fagerström Billai , BEA
Eukaryotic mRNAs are synthesized by the Pol II complex
Fredrik Fagerström Billai , BEA
Gene regulation in the eukaryotes
Wasserman and Sandelin (2004)
Fredrik Fagerström Billai , BEA
Chip- General outline
Fredrik Fagerström Billai , BEA
Chip analysis of ERα recruitment to estrogen responsive gene promoters.
Ray et al. 2006
Chip Quantification
Fredrik Fagerström Billai , BEA
Calella et al. 2007
Fredrik Fagerström Billai , BEA
Real Time PCR quantification
Fredrik Fagerström Billai , BEA
Real Time PCR quantification
Fredrik Fagerström Billai , BEA
Chip on Chip Outline
Fredrik Fagerström Billai , BEA
Genome Wide Chip Solutions
Affymetrix Tiling ArraysClassic Two Color Arrays Illumina Deep Sequencing
Fredrik Fagerström Billai , BEA
Millions of copies of a specificoligonucleotide probe
Single stranded, labeled ‘target’
Oligonucleotide ‘probe’
* **
**
1.28cm
GeneChip Probe Array
1.28cm
Affymetrix array architecture
Fredrik Fagerström Billai , BEABoyer et al. 2005
Tiling arrays provide high resolution for identifying bound fragments
Overlapping 25-mer fragments
Fredrik Fagerström Billai , BEA
Quantile Normalization
Fredrik Fagerström Billai , BEA
Identifying bound genomic fragments by Intensity Percentile Ranking.
Fredrik Fagerström Billai , BEA
Integrated Genome Browser, IGB
Fredrik Fagerström Billai , BEA
Genome-wide profiling of PRC1Polycomb chromatin binding in Drosophila melanogaster.
Fredrik Fagerström Billai , BEA
Chip profiles