Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HCT 116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared from 50 ng, 5 ng, or 0.5 ng enriched ChIP DNA or 50 ng Input DNA using SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Dual Index Primers) #47538, pooled into one sample, and sequenced on an Illumina® Next-Seq platform. The figure shows binding across GAPDH, a known target gene of H3K4me3. For additional ChIP-seq tracks, please download the product data sheet.
Learn more about how we get our imagesChromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HCT 116 cells and TCF4/TCF7L2 (C48H11) Rabbit mAb #2569, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared from 50 ng, 5 ng, or 0.5 ng enriched ChIP DNA or 50 ng Input DNA using SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Dual Index Primers) #47538, pooled into one sample, and sequenced on an Illumina® Next-Seq platform. The figure shows binding across CaMK2D, a known target gene of TCF4/TCF7L2. For additional ChIP-seq tracks, please download the product data sheet.
Learn more about how we get our imagesMetagene analysis of ChIP-seq data generated using different amounts of starting ChIP DNA. Analyses of both Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 (left) and TCF4/TCF7L2 (C48H11) Rabbit mAb #2569 (right) ChIP-seq data show that the signal-to-noise ratio for peaks identified across entire genome are similar for all three starting amounts of ChIP DNA.
Learn more about how we get our imagesNext generation sequencing (NG-seq) is a high throughput method that can be used downstream of chromatin immunoprecipitation (ChIP) assays to identify and quantify target DNA enrichment across the entire genome. The SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® contains all of the enzymes and buffers necessary to generate high quality DNA sequencing libraries from ChIP DNA for next-generation sequencing on the Illumina® platform. The fast, user-friendly workflow minimizes hands-on time needed for generation and purification of DNA libraries.
Each kit component must pass rigorous quality control standards, and for each new lot the entire set of reagents is functionally validated together by construction and sequencing of indexed libraries on the Illumina® sequencing platform.
This product must be used in combination with SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Single Index Primers) #29580 or SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Dual Index Primers) #47538. This product provides sufficient amounts of reagents for 24 reactions and is compatible with both enzymatic- and sonication-fragmented, ChIP-enriched DNA.
Compatible SimpleChIP® kits:
SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003
SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005
SimpleChIP® Plus Sonication Chromatin IP Kit #56383
SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Single Index Primers) #29580
SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Dual Index Primers) #47538
Non-Compatible SimpleChIP® kits:
SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads) #9002
SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads) #9004
Note: Agarose beads are blocked with sonicated salmon sperm DNA, which will contaminate DNA library preps and NG-seq.
Required Reagents
Reagents Included:
Reagents Not Included:
When performing ChIP-seq, it is necessary to generate a control DNA sequencing library that can be used to determine any experimental bias in DNA enrichment that is introduced during the ChIP assay and DNA library preparation. DNA purified from input chromatin (i.e. the chromatin that was used for the IP) is typically used to generate the control DNA library.
Starting Material: 500 pg –1 μg of ChIP DNA. To ensure optimal diversity of the DNA sequencing libraries, we recommend using 5 ng of ChIP-enriched DNA for transcription factor or co-factor ChIP-seq, 50 ng of ChIP-enriched DNA for total histone or histone modification ChIP-seq, and 50 ng of input DNA for the control DNA sequencing library. If necessary, less than 5 ng of ChIP-enriched DNA can be used for library generation; however, this may result in a lower diversity of library due to PCR bias during amplification.
Before starting:
Before starting:
Size selection is NOT recommended during the Cleanup Adaptor-ligated ChIP DNA phase, because it results in a dramatic decrease in both yield and diversity of ChIP-seq DNA libraries.
Before starting:
Before starting:
Reagents | Volume for 1 PCR Reaction (50 μl) |
Purified adaptor ligated ChIP-DNA fragments (from step 9, Section III) | 15 μl |
Q5® PCR Master Mix (•) | 25 μl |
Single Index Primer for Illumina® (•) (or Dual Index 7 Primer for Illumina® [orange cap] for dual indexing) | 5 μl |
Universal PCR Primer for Illumina® (•) (or Dual Index 5 Primer for Illumina® [white cap] for dual indexing) | 5 μl |
a. | Initial Denaturation | 98°C 30 sec |
b. | Denaturation | 98°C 10 sec |
c. | Anneal and Extension | 65°C 75 sec |
d. |
For starting material of 50 ng ChIP DNA, repeat steps b and c for a total of 6 cycles.
|
|
e. | Final Extension | 65°C 5 min |
f. | Hold | 4°C |
Before starting:
50 ng | 5 ng | 0.5 ng | 50 ng input DNA |
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Figure 1. Agilent Bioanalyzer® profiles of DNA libraries prepared from different starting amounts of ChIP DNA generated using the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared from 50 ng, 5 ng, and 0.5 ng of enriched ChIP DNA and 50 ng input DNA. As shown, each DNA library preparation shows a similar DNA fragment size (expected range 300 bp to 900 bp).
Libraries | Library Yield (ng) | Total Reads (Millions) | % Reads mapped to genome | Identified Peaks |
TCF4/TCF7L2 #2569 (50 ng) | 140.8 | 39.8 | 97.8 | 9425 |
TCF4/TCF7L2 #2569 (5 ng) | 187.8 | 34.4 | 97.8 | 10506 |
TCF4/TCF7L2 #2569 (0.5 ng) | 67.3 | 33.7 | 96.0 | 9416 |
H3K4me3 #9751 (50 ng) | 110.7 | 29.4 | 98.5 | 27967 |
H3K4me3 #9751 (5 ng) | 120.5 | 33.7 | 98.2 | 28665 |
H3K4me3 #9751 (0.5 ng) | 44.2 | 29.4 | 94.1 | 32221 |
Input DNA (50 ng) | 112.2 | 26.8 | 97.9 | N/A |
Table 1. Comparison of DNA sequencing libraries prepared from different starting amounts of ChIP DNA generated using the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. ChIP was performed using 4 x 106 HCT 116 cells and either TCF4/TCF7L2 (C48H11) Rabbit mAb #2569 or Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751. Libraries were generated using dual index primers provided in SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Dual Index Primers) #47538, pooled into one sample, and sequenced on an Illumina® Next-Seq platform. As shown, the number of identified peaks is similar for all three starting amounts of ChIP DNA.
Figure 2. ChIP-seq data generated using Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 and different starting amount of ChIP DNA, as described in Table 1. The figure shows binding across chromosome 12 (upper panel), including GAPDH (lower panel), a known target gene of H3K4me3. As shown, both peak patterns and peak heights are similar for all three starting amounts of ChIP DNA.
Figure 3. ChIP-seq data generated using TCF4/TCF7L2 (C48H11) Rabbit mAb #2569, as described in Table 1. The figure shows binding across chromosome 4 (upper panel), including CaMK2D (lower panel), a known target gene of TCF4/TCF7L2. As shown, both peak patterns and peak heights are similar for all three starting amounts of ChIP DNA.
Figure 4. Metagene analysis of ChIP-seq data generated using different amounts of starting ChIP DNA. Analyses of both Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 (left panel) and TCF4/TCF7L2 (C48H11) Rabbit mAb #2569 (right panel) ChIP-seq data show that the signal to noise ratio for peaks identified across entire genome are similar for all three starting amounts of ChIP DNA.
Description
End Prep Enzyme Mix is optimized to convert 500 pg-1 μg of fragmented DNA to repaired DNA having 5´-phosphorylated, 3´-dA-tailed ends.
Quality Control Assays
Quality Control Assays
Description
Ligation Master Mix is a ready-to-use solution of T4 DNA Ligase, proprietary ligation enhancer, and optimized reaction buffer.
Quality Control Assays
Efficiency (transformants/µg)
Recircularization | Insertion | |
Blunt ends | > 1 x 107 | > 2.5 x 106 |
Uncut vector | > 1 x 108 | N/A |
Quality Control Assays
Description
The Q5® PCR Master Mix (2X) is specifically optimized for robust, high fidelity amplification of next-generation sequencing (NGS) libraries, regardless of GC content. The polymerase component of the master mix, Q5 High-Fidelity DNA Polymerase, is a novel thermostable DNA polymerase that possesses 3´→5´ exonuclease activity, and is fused to a processivity-enhancing Sso7d domain. Q5 also has an ultra-low error rate (> 100-fold lower than that of Taq DNA Polymerase and ~12-fold lower than that of Pyrococcus furiosus (Pfu) DNA Polymerase). The buffer component of the master mix has been optimized for robust amplification, even with GC-rich amplicons and offers enhanced compatibility with a variety of beads used in typical NGS workflows. These features make the Q5® PCR Master Mix ideal for NGS library construction. This convenient 2X master mix contains dNTPs, Mg++ and a proprietary buffer, and requires only the addition of primers and DNA template for robust amplification. The inclusion of the hot start aptamer allows convenient room temperature reaction set up.
Quality Control Assays
posted November 2017
protocol id: 1624
Product Includes | Volume (with Count) | Storage Temp | |||
---|---|---|---|---|---|
End Prep Enzyme Mix | 1 x 72 µl | -20°C | |||
End Prep Reaction Buffer | 1 x 168 µl | -20°C | |||
Ligation Enhancer | 1 x 24 µl | -20°C | |||
Ligation Master Mix | 1 x 720 µl | -20°C | |||
Q5® PCR Master Mix | 1 x 600 µl | -20°C |
Next generation sequencing (NG-seq) is a high throughput method that can be used downstream of chromatin immunoprecipitation (ChIP) assays to identify and quantify target DNA enrichment across the entire genome. The SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® contains all of the enzymes and buffers necessary to generate high quality DNA sequencing libraries from ChIP DNA for next-generation sequencing on the Illumina® platform. The fast, user-friendly workflow minimizes hands-on time needed for generation and purification of DNA libraries. This product must be used in combination with SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Single Index Primers) #29580 or SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Dual Index Primers) #47538.
This product provides sufficient amounts of reagents for 24 reactions and is compatible with both enzymatic- and sonication-fragmented, ChIP-enriched DNA. This product is compatible with SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003, SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, and SimpleChIP® Plus Sonication Chromatin IP Kit #56383. This product is not compatible with SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads) #9002 and SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads) #9004 because agarose beads are blocked with sonicated salmon sperm DNA, which will contaminate DNA library preps and NG-seq.
This kit has been validated in combination with SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Single Index Primers) #29580 or SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Dual Index Primers) #47538 to generate qualified DNA libraries using 50 ng, 5 ng, or 0.5 ng ChIP DNA as starting materials. Libraries prepared from different starting amounts of ChIP DNA exhibit similar Bioanalyzer® profiles, genome mapping rates, numbers of identified binding peaks, and signal-to-noise ratios across the whole genome.
All Species Expected
The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This assay can be used to identify multiple proteins associated with a specific region of the genome, or the opposite, to identify the many regions of the genome bound by a particular protein (3-6). It can be used to determine the specific order of recruitment of various proteins to a gene promoter or to "measure" the relative amount of a particular histone modification across an entire gene locus (3,4). In addition to histone proteins, the ChIP assay can be used to analyze binding of transcription factors and co-factors, DNA replication factors and DNA repair proteins. When performing the ChIP assay, cells or tissues are first fixed with formaldehyde, a reversible protein-DNA cross-linking agent that "preserves" the protein-DNA interactions occurring in the cell (1,2). Cells are lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion. The chromatin is then immunoprecipitated with antibodies specific to a particular protein or histone modification. Any DNA sequences that are associated with the protein or histone modification of interest will co-precipitate as part of the cross-linked chromatin complex and the relative amount of that DNA sequence will be enriched by the immunoselection process. After immunoprecipitation, the protein-DNA cross-links are reversed and the DNA is purified. Standard PCR or Quantitative Real-Time PCR can be used to measure the amount of enrichment of a particular DNA sequence by a protein-specific immunoprecipitation (1,2). Alternatively, the ChIP assay can be combined with genomic tiling micro-array (ChIP on chip) techniques, high throughput sequencing, or cloning strategies, all of which allow for genome-wide analysis of protein-DNA interactions and histone modifications (5-8).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. SimpleChIP is a registered trademark of Cell Signaling Technology, Inc. AMPure is a registered trademark of Beckman Coulter, Inc. Bioanalyzer is a registered trademark of Agilent Technologies, Inc Illumina is a registered trademark of Illumina, Inc. Q5 is a registered trademark of New England Biolabs, Inc. SPRIselect is a registered trademark of Beckman Coulter, Inc. USER Enzyme is a registered trademark of New England Biolabs, Inc.
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Product # | Size | Price |
---|---|---|
56795S | 1 Kit (24 assays) | $ 700.0 |