Applications of ChIP. November 05, David Grotsky, PhD Scientific Support Specialist - Epigenetics

Transcription

1 Applications of ChIP November 05, 2014 David Grotsky, PhD Scientific Support Specialist - Epigenetics

2 Overview Introduction to chromatin and ChIP The histone code hypothesis What we learn from ChIP-on-chip What we learn from ChIP-seq Variations of ChIP 2

3 What is chromatin? 3

4 Histone modifications ac acetylation me methylation P phosphorylation cit deimination ub ubiquitination 4

5 ChIP overview Cross-link DNA and proteins (X-ChIP) Chromatin fragmentation by sonication (X-ChIP) or by enzymatic methods (micrococcal nuclease) (N-ChIP) Immunoprecipitation of the chromatin fragments interacting with the target protein/modification Reverse cross-link (X-ChIP) and DNA purification Downstream analysis Analysis of obtained material to determine abundance of target sequence(s) relative to input 5

6 An early link between histone acetylation and transcriptional activation Source: Hebbes et el, EMBO J,

7 An early link between histone acetylation and transcriptional activation Source: Hebbes et el, EMBO J,

8 The histone code hypothesis Source: Strahl and Allis, Nature,

9 Common methods of ChIP analysis ChIP-qPCR ChIP-on-chip ChIP-seq 9

10 An example of ChIP-qPCR 10

11 ChIP analysis genome-wide Chip-on-chip 11

12 Acetylation and methylation genome wide Source: Bernstein et al., PNAS,

13 ChIP-on-chip validation Source: Bernstein et al., PNAS,

14 Using ChIP-on-chip to identify promoters Source: Kim et al., Nature,

15 Using ChIP-on-chip to identify promoters Source: Kim et al., Nature,

16 Using ChIP-on-chip to identify promoters Source: Kim et al., Nature,

17 Histone modification map of promoters Source: Heintzman et al., Nature Genetics,

18 Histone modification map of enhancers Source: Heintzman et al., Nature Genetics,

19 Limitations of ChIP-on-chip Requires large cell numbers Not sensitive to repetitive elements Large number of arrays are necessary to cover entire genome Amplification bias Source: Bernstein et al., Cell,

20 ChIP-seq Source: Barski et al., Cell,

21 Higher resolution with ChIP-seq Source: Park, Nature Reviews Genetics,

22 Higher resolution histone modification map Source: Barski et al., Cell,

23 Higher resolution histone modification map Source: Barski et al., Cell,

24 Dashboard of histone modifications Source: Zhou et al., Nature Reviews Genetics,

25 Histone modifications on an active gene Source: Barth and Imhof, Trends in Biochemical Sciences,

26 The ENCODE Project ENCyclopedia Of DNA Elements Identify all functional elements in the human genome UCSC Genome Browser: 26

27 ChIP-seq guidelines Antibody validation Sequencing depth Experimental reproducibility Data quality control Data reporting Source: Landt, et al., Genome Research,

28 Variations of ChIP ChIP with low cell numbers ChIP in under 5 hours ChIP-loop Adds an IP step to chromatin conformation capture (3C) identifies long range DNA interactions Detects DNA-DNA interactions mediated by a specific protein of interest ChIA-PET (Chromatin Interaction Analysis by Paired-End Tag Sequencing) Genome wide ChIP-loop During ligation, linker sequences are added to create a library of interacting regions 28

29 Variations of ChIP ChIP-exo ChIP-BSseq Detects protein binding at single-nucleotide resolution DNA not bound directly by protein is removed PCR adapters are added to ChIP d DNA and DNA is digested a 5 3 exonuclease Crosslinks are reversed and DNA is amplified using primers to the first adapter Second adapter is added to 5 ends and product is sequenced Combination of ChIP and bisulfite sequencing ChIP products are bisulfite treated and then sequenced 29

30 Products for your research

31 Characteristics of abcam ChIP kits Simple to use Reaction takes place in the wells of the 96-wp: easy for standardization and HTP Quick Only 5 hours to perform ChIP (compared to 2 days using conventional method) Inclusive Kit contains all necessary reagents for ChIP reaction (excluding cross-linking step) Compatible Eluted DNA can be processed straight away by any DNAamplification method 31

32 New ChIP kit High Sensitivity (ab185913) Non-Immune IgG RNA polymerase II antibody Relative enrichment fold Low cell input: 2000 cells or 0.5mg of tissue per prep Protocol time: 5hrs (chromatin to DNA) Convenient format: 96 well plate format 1 0 2,000 4,000 20, , ,000 Cell number 32

33 MeDIP (methylated DNA ChIP) Kits Specific antibody to enrich 5-mC/5-hmC rich DNA regions MeDIP Detects 5-mC (methylated cytosine) residues hmedip Detects 5-hmC (hydroxymethylated cytosine). 5-hmC is a recently discovered modification in animal cells apparently involved in DNA demethylation, but its role is not clear Ready in less than 4 hours 33

34 Chromatin Extraction Kit (ab117152) Optimized kit to prepare chromatin for ChIP experiments Processing time < 1 hour Sample: 10 5 cells or 10 mg tissue Chromatin extracted from 3x10 6 mouse neuroblastoma cells. Yield = 4 µg chromatin/10 6 cells Source: Image courtesy of anonymous Abreview 34

35 Webinar promotion As a thank you Save 20% on ChIP kits Hurry! This promotion is for a limited period only Simply quote the promo-code below at purchase APPCHIP-XBLXZ

36 Did you know Abcam has a technical webinar series? Archived Webinars include ChIP ELISA In-Cell ELISA Western Blot Immunohistochemistry Immunocytochemistry Immunoprecipitation In-Cell ELISA Flow Cytometry Post Translations Modifications Have questions in regards to techniques? All of our past technical webinars are available online. Watch the webinars within your own lab, on your time Visit Abcam.com/events homepage for a full list of upcoming webinars and events 36

37 Functions of Non-Coding RNAs in Evolution, Epigenetics and Therapeutic Applications January 15, 2015 Singapore Free meeting to attend Early Registration Required Meeting organizers: Prof. Lawrence Stanton & Prof. Michael Rossbach, Genome Institute of Singapore Keynote Speaker: John Mattick, Garvan Institute, AU "RNA at the epicenter of human development Confirmed Speakers Timothy Bredy, The University of Queensland, Brisbane, QLD, Australia Roger Foo, National University Hospital, Singapore Michael Rossbach, GIS, Singapore Prabha Sampath, IMB, Singapore Lawrence Stanton, GIS, Singapore Leah Vardy, IMB, Singapore Wan Yue, GIS, Singapore Speaker talk titles and more information at 37

38 Frontiers of Cell Signaling June 21-24, 2015 Shanghai, China Prof Yimin Zou, University of California - San Diego,US Yingzi Yang, NIH, US Dan Wu, Yale University, US Peter Lawrence, University of Cambridge, UK Andy McMahon, University of South California, US Susan Taylor, UCSD, US Early bird registrations: April 17, 2015 (save up to $325!) Oral abstract submission: April 17, 2015 Standard registrations: May 7, 2015 Poster abstract submission: May 7, 2015 Speaker talk titles and more information at

39 Questions? 39