obtain the DNA methylation profile of your ChIP-enriched DNA
Active Motif's ChIP-Bisulfite-Sequencing (ChIP-Bis-Seq) Kit combines the target-specific selection of chromatin immunoprecipitation (ChIP) with bisulfite conversion and sequencing (Bis-Seq) to provide single nucleotide resolution of immunoprecipitated DNA. By combining the two techniques, information about both the chromatin context and the methylation profiles of each DNA strand can be evaluated, enabling better understanding of the biological context and significance regarding gene regulation, gene expression, mechanisms of chromatin modification and pathway analysis.
Complete validated workflow for performing ChIP-Bis-Seq.
The ChIP-Bis-Seq Kit provides a complete and validated workflow that includes chromatin preparation and immunoprecipitation reagents, methylated adapters, sequencing library reagents and bisulfite conversion components. The ChIP-Bis-Seq Kit is compatible with Illumina® sequencing platforms.
- Directly interrogate both chromatin and DNA methylation profiles on the same sample with higher resolution than either ChIP or bisulfite sequencing alone
- Evaluate allele-specific differences in DNA (imprinting, X-inactivation)
- Method works with histone and transcription factor antibodies
- Optimized ChIP reagents reduce background caused by non-specific binding and provide higher quality DNA for analysis
- Reduce sequencing costs by focusing on specific genomic regions of interest
- Save time and money by using our already validated ChIP-Bis-Seq workflow
|ChIP-Bis-Seq Kit||10 libraries||53048||$895||Buy Now|
|High Sensitivity Chromatin Preparation||16 rxns||53046||$255||Buy Now|
|ChIP-Bis-Seq Kit Manual|
|High Sensitivity Chromatin Preparation Kit Manual|
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How does the ChIP-Bis-Seq Kit work?
Figure 1: Flow chart of the ChIP-Bis-Seq method.
High Sensitivity ChIP Reagents
The ChIP-Bis-Seq Kit includes Active Motif's ChIP-IT High Sensitivity® Kit for the chromatin preparation and immunoprecipitation reactions. The ChIP-IT High Sensitivity Kit is ideal for use with challenging antibodies that do not give signal with other ChIP methods, as the method is sensitive enough to detect specific binding of even low abundance proteins. Below is a comparison of ChIP-IT High Sensitivity with other commercially available ChIP kits. ChIP was performed using untreated MCF-7 chromatin and an antibody that binds the low abundance nuclear receptor co-activator 2 (NCOA2) transcriptional co-factor. It was also performed with a negative control IgG and a negative control primer set (Active Motif Catalog No. 71001) to monitor non-specific binding. Only the ChIP-IT High Sensitivity method was able to detect NCOA2 binding at specific genomic locations (ESRRA promoter), providing approximately 20-fold higher enrichment than the negative controls. Although ESRRA shows qPCR signals using the kit from Competitor D, the enrichment levels are poor due to the high background from the negative control primer set and the non-specific binding detected with the IgG.
Figure 1: Comparison of ChIP kits targeting a low abundance transcription factor.
Efficient Bisulfite Conversion
The ChIP-Bis-Seq Kit utilizes Active Motif's highly efficient bisulfite conversion kit. Following ChIP enrichment, the DNA undergoes end repair and the addition of A overhangs to the 3´ends of DNA fragments. Methylated adapters (compatible with Illumina seqeuncing platforms) are included in the ChIP-Bis-Seq Kit for ligation to the ChIP DNA. The methylated adapters contain methylated cytosine residues throughout the adapter sequence. This prevents conversion of the cytosines during the bisulfite conversion tratement, thereby preserving the original adapter sequence for downstream PCR amplification. During bisulfite conversion, methylated cytosines will be preserved and non-methylated cytosines will be converted into uracil with >98% efficiency. Finally, the ChIP-Bis-Seq Kit contains specialized desulfonation and purification columns that are ideal for use with bisulfite-converted, fragmented, ChIP DNA.
Figure 2: Bisulfite sequencing results of PC9 cells.
Multiple Parameter Analysis
The advantage of the ChIP-BIs-Seq Kit is that it enables direct interrogation of both chromatin and DNA methylation profiles on the same sample. It combines the enrichment properties of chromatin immunoprecipitation to look at specific protein/DNA binding interactions with the single-base resolution of bisulfite sequencing to determine the methylation profile of the DNA binding site. It may be used to evaluate allele-specific differences in DNA methylation (imprinting, X-inactivation) or to study gene regulatory effects.
Figure 3: ChIP-Bis-Seq sequencing and DNA methylation results.
Contents & Storage
The ChIP-Bis-Seq Kit contains separate modules for each stage of the process. One module is for chromatin preparation and immunoprecipitation, another module contains Next-Generation sequencing library reagents and a third module includes the reagents needed for bisulfite conversion. Reagents within each module may contain multiple storage temperatures. All components can be stored at -20°C prior to first use, then we recommend storing each component at the temperatures indicated below. Do not re-freeze the Protein G Agarose Beads once they have been thawed for use.
Reagents for Chromatin Preparation and Immunoprecipitation (16 reactions)
- RNase A (10 µg/µl); Store at -20°C
- Proteinase K (10 µg/µl); Store at -20°C
- Blocker; Store at -20°C
- 100 mM PMSF; Store at -20°C
- Protease Inhibitor Cocktail (PIC); Store at -20°C
- Precipitation Buffer; Store at -20°C
- Carrier; Store at -20°C
- 10X PBS; Store at -20°C
- Fixation Buffer; Store at 4°C
- Protein G Agarose Beads; Store at 4°C
- TE, pH 8.0; Store at RT
- Detergent; Store at RT
- Stop Solution; Store at RT
- Chromatin prep Buffer; Store at RT
- ChIP Filtration Columns; Store at RT
- ChIP Buffer; Store at RT
- 5 M NaCl; Store at RT
- Wash Buffer AM1; Store at RT
- Elution Buffer AM4; Store at RT
- DNA Purification Binding Buffer; Store at RT
- 3 M Sodium Acetate; Store at RT
- DNA Purification Wash Buffer; Store at RT
- DNA Purification Elution Buffer; Store at RT
- DNA Purification Columns; Store at RT
Reagents for Sequencing Library Preparation (10 libraries)
- 10X Reaction Buffer AM3; Store at -20°C
- 2X Quick Ligation Buffer; Store at -20°C
- 10X T4 DNA Ligase Buffer; Store at -20°C
- DNA Polymerase I Klenow (5 U/µl); Store at -20°C
- dNTP Mix, 10 mM each; Store at -20°C
- Klenow Fragment (3´ -5´ exo-) (5 U/µl); Store at -20°C
- T4 DNA Ligase (2,000 U/µl); Store at -20°C
- T4 DNA Polymerase (3 U/µl); Store at -20°C
- T4 Polynucleotide Kinase; Store at -20°C
- 1 mM dATP; Store at -20°C
- Methylated adapters (5 µM); Store at -20°C
- Glycogen; Store at -20°C
Reagents for Bisulfite Conversion (10 conversions)
- Conversion Reagent; Store at RT
- Hydroquinone; Store at RT
- Buffer A; Store at RT
- Buffer B; Store at RT
- DNA Binding Buffer; Store at RT
- DNA Wash Buffer; Store at RT
- DNA Elution Buffer; Store at RT
- ChIP-Bis-Seq purification columns & collection tubes; Store at RT