Low Cell ChIP-Seq Kit
complete ChIP-Seq workflow from limited cell numbers
Active Motif's Low Cell ChIP-Seq Kit provides a complete ChIP-Seq workflow for chromatin preparation, immunoprecipitation and NGS library preparation from limited amounts of cells or small tissue biopsies.
Cell Number Recommendations.
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The assay is robust enough to work with both high abundance histone modifications and low abundance transcription factor proteins. The Next Gen DNA Library and Next Gen DNA Indexing reagents are included to generate high complexity sequencing libraries that are compatible with Illumina® platforms.
ChIP-Seq is a method that has traditionally relied on using millions of cells per immunoprecipitation reaction to provide information about global changes across large populations of cells. The Low Cell ChIP-Seq Kit not only reduces sample input requirements, but it also resolves issues often associated with low-cell ChIP, including poor signal-to-noise, inefficent library amplification and high duplication rates.
Low Cell ChIP-Seq Kit Highlights
- Reproducible ChIP-Seq data from as little as 1,000 to 50,000 cells depending on protein abundance
- Complete ChIP-Seq workflow includes chromatin preparation, immunoprecipitation and Illumina-compatible NGS library preparation reagents that have been optimized for use on limited cell numbers
- Profile histone or transcription factor DNA binding sites
- Blocking reagents and an optimized protocol improve signal-to-noise ratio for better peak calling and lower background
- Molecular identifiers (MIDs) are incorporated within the P5 adapter for retention of fragmentation duplicates and removal of PCR duplicates to increase the number of unique alignments
- Multiplex up to 16 samples on the same sequencing flow cell
The Low Cell ChIP-Seq Kit contains enough reagents to perform 16 ChIP-seq reactions. To learn more about Low Cell ChIP-Seq, click on the Data, or Contents tabs below.
What is Low Cell Number?
With Low Cell ChIP-Seq, researchers can reduce the number of cells they are studying to improve their understanding of the complexities of protein-DNA interactions to a more limited population of cells. The assay is ideal if working with limited sample amounts, hard to culture cell lines, or small tissue biopsies. The recommended cell number is the amount of cells that will be used for each chromatin preparation. The entire chromatin sample is then used in a single immunoprecipitation reaction. This differs from other ChIP assays where chromatin must be prepared from larger cell numbers and then the chromatin is diluted for use in the immunoprecipitation.
Figure 1: Table of recommended cell numbers for use with Low Cell ChiP-Seq Kit based on target protein abundance.
Other factors that will influence the recommended cell number include cell type, abundance of target protein and the quality of the ChIP-Seq antibody. Below are some validated antibodies from Active Motif for use with Low Cell ChIP-Seq.
|Protein Target||Active Motif Catalog Number||Cell Type Tested|
Figure 2: Table of Active Motif's Low Cell ChIP-Seq validated antibodies.
To determine protein abundance within your sample type, it may be necessary to evaluate expression data, research protein databases, or assay a variety of cell numbers to determine the optimal amount needed. Below is a graph of data generated using Active Motif's Histone H3 PTM Multiplex Assay (Catalog No. 33115). This assay evaluates up to 14 histone modifications within the same sample well. Various mouse tissues were analyzed to determine the relative abundance of histone modifications across sample types. Data shows some histone modifications, such as H3K27ac and H3K56ac, display low abundance across all sample types tested. However, other modifications, such as H3K27me2 and H3K9me3, show tissue-specific differences in the levels of the histone modification.
Figure 3: Histone H3 PTM Multiplex Kit (Catalog No. 33115) data of Histone Abundance in Mouse Tissue.
Low Cell ChIP-Seq Data
Active Motif's Low Cell ChIP-Seq Kit was used to immunoprecipitate 10,000 MDA-MB-468 cells using an antibody directed against Bromodomain containing 4 (BRD4) protein, or 5,000 GM12878 cells using antibodies directed against Histone H3K27ac or Histone H3K27me3. Following ChIP, Illumina-compatible sequencing libraries were prepared using the included Next Gen DNA Library Kit and Next Gen Indexing Kits and sequenced using the NextSeq 500. Results show high quality ChIP-seq peaks can be obtainined using as little as 5,000 cells for both active and repressive histone modifications and as low as 10,000 cells for BRD4. Peaks were compared to ENCODE data sets using 20 million cells.
Figure 4: Low Cell ChIP-Seq peaks for BRD4, H3K27ac and H3K27me3 histone modifications compared to ENCODE data sets.
Below is a summary of Low Cell ChIP-Seq data sets using both robust and low abundance target proteins. Nice peaks above background were observed from as little as 1,000 cells for histone marks H3K4me3, H3K4me1 and H3K27me3. Both active and repressive histone modifications are shown, indicating that the method is sensitive enough to obtain good ChIP-Seq signal even when working with repressive chromatin. Lower abundance proteins, including BRD4 and insulator protein CTCF showed nice ChIP enrichment from as low as 10,000 cells. Results shown are from a single Low Cell ChIP-Seq reaction, however, data has been reproducibly generated across multiple experiments.
Figure 5: Summary of various Low Cell ChIP-Seq experiments targeting both histone and low abundance proteins.
Contents & Storage
Please note that the Low Cell ChIP-Seq Kits are shipped on dry ice and contains reagents with multiple storage temperatures inside. All components can be stored at -20°C prior to first use, then we recommend storing each component at the temperatures indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. Each kit includes the following components:
Reagents for Low Cell ChIP-Seq Kit
- 10X PBS; Store at -20°C
- Proteinase K (10 µg/µl); Store at -20°C
- Blocker; Store at -20°C
- Blocking Reagent AM1; Store at -20°C
- BSA; Store at -20°C
- 100 mM PMSF; Store at -20°C
- Protease Inhibitor Cocktail (PIC); Store at -20°C
- Carrier; 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
- 5 M NaCl; Store at RT
- Stop Solution; Store at RT
- ChIP Filtration Columns; Store at RT
- ChIP Buffer; Store at RT
- Wash Buffer AM1; Store at RT
- Elution Buffer AM4; Store at RT
- LiCl Buffer; Store at RT
- 1.7 ml siliconized tubes; Store at RT
Reagents for Next Gen DNA Library Kit* (included)
- Buffer W1; Store at -20°C
- Enzyme W2; Store at -20°C
- Buffer G1; Store at -20°C
- Reagent G2; Store at -20°C
- Enzyme G3; Store at -20°C
- Enzyme G4; Store at -20°C
- Buffer Y1; Store at -20°C
- Enzyme Y3 Store at -20°C
- Buffer B1; Store at -20°C
- Reagent B3; Store at -20°C
- Enzyme B4; Store at -20°C
- Enzyme B5; Store at -20°C
- Enzymbe B6; Store at -20°C
- Reagent R2; Store at -20°C
- Buffer R3; Store at -20°C
- Enzyme R4; Store at -20°C
- Alu 115; Store at -20°C
- Alu 247; Store at -20°C
- PEG Solution; Store at RT
- Low EDTA TE; Store at RT
Reagents for Next Gen Indexing Kit* (included)
- Reagent Y2 Index 1; Store at -20°C
- Reagent Y2 Index 2; Store at -20°C
- Reagent Y2 Index 3; Store at -20°C
- Reagent Y2 Index 4; Store at -20°C
- Reagent Y2 Index 5; Store at -20°C
- Reagent Y2 Index 6; Store at -20°C
- Reagent Y2 Index 7; Store at -20°C
- Reagent Y2 Index 8; Store at -20°C
- Reagent Y2 Index 9; Store at -20°C
- Reagent Y2 Index 12; Store at -20°C
- Reagent Y2 Index 13; Store at -20°C
- Reagent Y2 Index 14; Store at -20°C
- Reagent Y2 Index 15; Store at -20°C
- Reagent Y2 Index 16; Store at -20°C
- Reagent Y2 Index 18; Store at -20°C
- Reagent Y2 Index 19; Store at -20°C
- Reagent R1; Store at -20°C
- Reagent B2 MID; Store at -20°C
* The Next Gen DNA Library and Next Gen Indexing Kits are powered by Swift Biosciences.