Active Motif,
Tools to analyze nuclear function,
Your CartYour Cart 0 items

Low Cell ChIP-Seq Kit

限られた細胞数からの完全なChIP-Seqワークフロー

アクティブ・モティフの Low Cell ChIP-Seq Kit 、限られた数の細胞、または少量の組織生検から、クロマチン調製、免疫沈降、およびNGSライブラリー調製のための完全なChIP-Seqワークフローを提供します。

Table of Low Cell Number recommendations.
推奨細胞数

このアッセイは、多量に存在するヒストン修飾だけでなく、少量しか存在しない転写因子タンパク質でもワークします。Illumina®プラットフォームと互換性がある複雑性の高いシーケンシグライブラリーを作製するために、Next Gen DNA LibraryNext Gen DNA Indexing の試薬がこのキットには含まれています。

ChIP-Seqは、細胞全体にわたるグローバルな変化についての情報を与えるために、通常数百万の細胞を用いて免疫沈降反応を行う実験手法です。Low Cell ChIP-Seq Kitはサンプルインプットの必要量を減少させるだけでなく、少量細胞ChIPでしばしば起こる不十分なS/N比、非効率なライブラリー増幅、高重複率の問題を解決します。

 

Low Cell ChIP-Seq Kit の特長

  • タンパク質存在量に応じて、わずか1,000個から50,000個の細胞で再現可能なChIP-Seqデータを得られます。
  • 完全なChIP-Seqワークフローは、限られた細胞数で使用できるようにクロマチン調製、免疫沈降、Illumina互換性NGSライブラリー調製用の試薬が最適化されています。

  • DNA結合サイトにおけるヒストンや転写因子のプロファイルができます。

  • ブロッキング試薬と最適化されたプロトコルにより、より良いpeak callingと低バックグラウンドが得られるため、S/N比が改善されます。
  • 分子識別(MID)は、ユニークアライメントの数を増加させるために、フラグメント化重複の維持やPCR重複の除去のために、P5アダプター内に組み込まれています。

  • 同じシーケンシングフローセル上に16サンプルまでマルチプレックスができます。

Low Cell ChIP-Seq Kit 16回のChIP-Seq反応を行うことが可能です。Low Cell ChIP-Seqについての詳細は、下のDataもしくは Contentsタブをクリックしてください。

 
Name Format Cat No. 価格 (税抜)  
Low Cell ChIP-Seq Kit 16 rxns 53084 ¥220,000 Buy Now
ChIP Buffer 50 ml 37516 ¥9,200 Buy Now
Blocking Reagent AM1 0.1 ml 37496 ¥7,800 Buy Now
BSA (10 mg/ml) 0.1 ml 37497 ¥10,600 Buy Now
Blocker 0.1 ml 37498 ¥13,000 Buy Now
Protein G Agarose Beads 1.2 ml 37499 ¥20,000 Buy Now
TE, pH 8.0 35 ml 37515 ¥7,800 Buy Now

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.

Table of Recommended Cell Numbers.
Figure 1: Table of the minimum recommended cell numbers for use with Low Cell ChIP-Seq Kit. Minimum cell numbers suggested are based on target protein abundance and antibody affinity.

 

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
Low Cell ChIP-Seq Validated Antibodies
H3K4me1 39297 GM12878
H3K4me3 39159  MDA-MB-468; GM12878
H3K9ac 39918  GM12878
H3K9me3 39161  GM12878
H3K27ac 39133  HepG2
H3K27me3 39155  MDA-MB-468
CTCF 61311  MDA-MB-468
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 PTM Multiplex Service. 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.

Histone H3 PTM Multiplex Data of Histone Abundance in Mouse Tissue.
Figure 3: Histone H3 PTM Multiplex 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.

Histone H3 PTM Multiplex Data of Histone Abundance in Mouse Tissue.
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.

Histone H3 PTM Multiplex Data of Histone Abundance in Mouse Tissue.
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
  • Enzyme 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.