Chromatin immunoprecipitation (ChIP) sequencing
Using the SOLiD® Platform for ChIP-Seq Analysis
The SOLiD® ChIP-Seq Kit is an optimized system for genome-wide ChIP analysis on SOLiD® sequencers, which includes ChIP preparation reagents and library generation reagents for 20 samples along with a unique optimized protocol. The simplified workflow involves cross-linking of proteins, cell lysis, and subsequent chromatin shearing and immunoprecipation utilizing Dynabeads®. The result is purified DNA which can be utilized for DNA fragment library construction utilizing best-in class reagents from the SOLiD® Sequencing system.
SOLiD® ChIP-Seq Kit workflow
Advantages of the SOLiD® ChIP-Seq Kit
The kit offers an optimized workflow which not only gives excellent sequencing results but also allows you to save an entired day compared to traditional ChIP protocols (see table 1).
Reliable purification--improved reproducibility due to optimized magnetic DNA purification
Higher sensitivity--allows the use of fewer cells per ChIP experiment to minimize variability betweene experiments
Lower input DNA--required--library preparation with reduced amount of DNA from 1-10 ng
Table 1. Comparison to a conventional ChIP protocol.
|Workflow step||MAGnify™ ChIP timeline||Conventional ChIP timeline|
|Antibody/chromatin incubation||2 hr||Overnight|
|Bead pulldown||1 hr||2 hr|
|Washes||30 min (2 buffers)||1- 3 hr (4 buffers)|
|Reverse crosslinking||1.5 hr||Overnight|
|Proteinase K digestion||2 hr|
|DNA elution from beads||15-30 min|
|DNA purification||2 hr- overnight|
|Average time||5 hours||36-48 hours|
Figure 1. Titration of cell number with MAGnify™ ChIP.
Sheared chromatin from MCF7 cells was prepared from 1 million cells in 50 μl lysis buffer and diluted to indicated number of cells per ChIP according to the MAGnify™ ChIP protocol. 1 μg of antibody (IgG–included in kit, H3-K27Me3–Cat. no. 49-1014, H3-K9Ac–Cat. no. 49-1009) were used for each ChIP experiment. 1% of the sonicated chromatin was set aside as input control. Optimized qPCR primers were used to amplify the SAT2 locus (Cat. no. 49-2026). Data are graphed as percent input.
Figure 2. 10,000 cell input with MAGnify™ ChIP.
Sheared chromatin from MCF7 or MDA-MB231 cells was prepared from 1 million cells in 50 μl lysis buffer and diluted to 10,000 cells per ChIP, according to the MAGnify™ ChIP protocol. 1 μg of antibody (IgG—included in kit, H3-K27Me3—Cat. no. 49-1014, H3-K9Ac—Cat. no. 49-1009) or 3μl RNA pol II (Cat. no. 49-1033) were used for each ChIP experiment. 1% of the sonicated chromatin was set aside as input control. Optimized qPCR primers were used to amplify to different loci: RARb1 promoter (Cat. no. 49-2027) or ER-a promoter (Cat. no. 49-2028). Data are graphed as percent input.
Figure 3. MAGnify™ ChIP versus conventional protocols.
Sheared chromatin from 293Gt cells was prepared and 50,000 cells used for ChIP (MAGnify™ ChIP) and 1 million cells per ChIP used for the conventional ChIP protocol. 1 μg of antibody (IgG— included in kit, H3-K9Me3—Cat. no. 49-1008) was used for each ChIP experiment. 1% of the sonicated chromatin was set aside as input control. Optimized qPCR primers were used to amplify the SAT2 locus (Cat. no. 49-2026), a known target of heterochromatin-associated H3-K9Me3. Key elements of a conventional protocol use an overnight immunoprecipitation step of antibody and chromatin, followed by enrichment with Protein A sepharose beads and extensive washes with buffers containing variable salt concentrations. Reverse crosslinking was done overnight at 65°C and DNA purification performed by phenol-chloroform extraction.