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ab185908 is a complete set of reagents required for carrying out a successful ChIP-Seq starting from mammalian cells or tissues. The kit is designed to selectively enrich a chromatin fraction containing specific DNA sequences from various species, particularly mammals, and to prepare a ChIP-Seq library for next generation sequencing using Illumina® platforms such as Illumina® Genome Analyzer II, HiSeq and MiSeq systems. The optimized protocol and components of the kit allow capture of low abundance protein/DNA complexes with minimized non-specific background levels and the ability to construct both non-barcoded (singleplexed) and barcoded (multiplexed) ChIP-Seq libraries quickly with reduced bias.
Starting materials can include various tissue or cell samples such as culture cells from a flask or plate, fresh and frozen tissues, etc.
Input Amount of Tissue/Cells:
In general, the amount of cells and tissues for each reaction can be 1 x 105 to 1 x 106 and 5 mg to 50 mg, respectively. For optimal preparation, the input amount should be 4 to 5 x 105cells or 20 to 30 mg tissues so that the amount of DNA enriched from the ChIP reaction can range from at least 1 ng to 100 ng.
Illumina® is a registered trademark of Illumina, Inc.
Protein-DNA interaction plays a critical role for cellular functions such as signal transduction, gene transcription, chromosome segregation, DNA replication and recombination, and epigenetic silencing. Identifying the genetic targets of DNA binding proteins and knowing the mechanisms of protein-DNA interaction on a genome-wide scale is important for understanding cellular processes. Chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) offers an advantageous tool for studying genome-wide protein-DNA interactions. It allows for detection that a specific protein binds to specific sequences in living cells. In particular, ChIP antibodies targeted against various transcriptional factors (TF) for genome-wide transcription factor binding site analysis by Chip-Seq is in high demand. Such analysis requires that ChIPed DNA contain minimal background for reliably identifying true TF-enriched regions. Currently used ChIP-Seq methods play an important role in identifying genome-wide protein-DNA interaction. The ChIP-Seq High Sensitivity Kit combines microplate-based ultra ChIP and high sensitive DNA library construction technologies.
|Components||12 tests||24 tests|
|1000X Protease Inhibitor Cocktail||1 x 15µl||1 x 30µl|
|10X End Polishing Buffer||1 x 30µl||1 x 60µl|
|2X HiFi PCR Master Mix||1 x 160µl||1 x 320µl|
|2X Ligation Buffer||1 x 250µl||1 x 500µl|
|8-Well Assay Strips (with Frame)||1 x 2 units||1 x 4 units|
|8-Well Strip Caps||1 x 2 units||1 x 4 units|
|Adaptors (50 μM)||1 x 15µl||1 x 30µl|
|Adhesive 8-Well Strip Film||1 x 4 units||1 x 8 units|
|Antibody Buffer||1 x 1ml||1 x 2ml|
|Anti-RNA Polymerase II||1 x 5µl||1 x 10µl|
|Blocker Solution||1 x 1ml||1 x 2ml|
|ChIP Buffer||1 x 6ml||1 x 12ml|
|DNA Binding Solution||1 x 7ml||1 x 14ml|
|DNA Elution Buffer||1 x 0.5ml||1 x 1ml|
|DNA Release Buffer||1 x 7ml||1 x 14ml|
|Elution Buffer||1 x 1ml||1 x 2ml|
|End Polishing Enhancer||1 x 13µl||1 x 26µl|
|End Polishing Enzyme Mix||1 x 13µl||1 x 26µl|
|Enrichment Enhancer||1 x 25µl||1 x 50µl|
|F-Collection Tube||1 x 15 units||1 x 30 units|
|F-Spin Column||1 x 15 units||1 x 30 units|
|GAPDH Primer - Forward (20 µM)||1 x 5µl||1 x 10µl|
|GAPDH Primer - Reverse (20 µM)||1 x 5µl||1 x 10µl|
|Lysis Buffer||1 x 7ml||1 x 14ml|
|MQ Binding Beads||1 x 1.6ml||2 x 1.6ml|
|Non-Immune IgG (1 mg/ml)||1 x 5µl||1 x 10µl|
|Primer I (10 μM)||1 x 15µl||1 x 30µl|
|Primer U (10 μM)||1 x 15µl||1 x 30µl|
|Proteinase K (10 mg/mL)||1 x 30µl||1 x 60µl|
|Rnase A||1 x 15µl||1 x 30µl|
|T4 DNA Ligase||1 x 15µl||1 x 30µl|
|Wash Buffer||1 x 12ml||1 x 25ml|
Our Abpromise guarantee covers the use of ab185908 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|CHIPseq||Use at an assay dependent concentration.|
Ten nanograms of DNA was ChIPed by RNA polymerase II enrichment and used for DNA library preparation.
The sheared chromatin isolated from different number of MBD-231 cells was used for ChIP-qPCR analysis of RNA polymerase II enrichment in GAPDH promoters.
A ChIP-sequencing library was prepared using ChIP-Seq High Sensitivity Kit (ab185908) from rat heart chromatin and polyclonal antibodies against H3K4me3, H3(K9/14)ac and, H3K27me3. Sequencing was carried out on an Illumina HiSeq2500. Bioinformatics analysis of ChIP-seq is performed utilizing Bowtie and MACS.
ab185908 has not yet been referenced specifically in any publications.