ChIP-seq on 10.000 cells

Robust automated or manual ChIP and library prep kits

  • Revolutionary: Only 10,000 cells needed for complete ChIP-seq procedure
  • Reliable and accurate sequencing library preparation from just picogram inputs
  • Simple 3-step library prep protocol with NO intermediate purification steps
  • Validated on studies for histone marks and with the IP-Star® Compact Automated Platform
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The Diagenode MicroPlex kit is the quickest and most efficient way to make sequencing libraries, especially from samples with very low inputs.  We regularly start with picogram amounts of ChIP material and produce excellent quality libraries that would be impossible to make using normal methods. Sequencing libraries made from the MicroPlex kit give us excellent results even in large genomes. The kit performs very well, and we will use the kit in the future for studies with low cell numbers or starting material.

Dr. Morgan Sammons, Lab of Dr. Shelley Berger, University of Pennsylvania

 

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High reproducibility ChIP on 10,000 cells with the IP-Star® Compact

ChIP was performed with IP-Star® Compact on human HeLa cells using the Diagenode antibody H3K4me3 (Cat No. pAb-003-050). Sheared chromatin from 10 000 cells, 0.25 µg of the H3K4me3 antibody and 0.25 μg of the negative IgG control were used per IP. Quantitative PCR was performed with the positive controls GAPDH-TSS and EIF4A2 promoter and the negative controls Myoglobin exon 2 and Sat 2 primer sets. The recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis) is shown in this figure.

Average and error bars of 10 IP's performed with IP-Star® Compact on Human Hela cells using the Diagenode antibody H3K4me3.

Reliable detection of enrichments in ChIP-seq

ChIP was peformed with H3K4me3 antibody, 17 pg of DNA (subsequently amplified), ChIP'd from 10,000 cells and 35 pg of control DNA (subsequently amplified), ChIP'd from 100,000 cells. The IP'd DNA was amplified and transformed into a sequencing-ready preparation for the Illumina platform with the True MicroAmplification kit. The library was analyzed on an Illumina Genome Analyzer. Cluster generation and sequencing were performed using manufacturer instructions.

Perfect match with ChIP-seq reference dataset

 

  Matched by Broad Institute dataset
  Unmatched by Broad Institute dataset

We observed a perfect match between the top 40% of True MicroChIP peaks and the reference dataset. Based on the NIH Encode project criterion, ChIP-seq results are considered reproducible between an original and reproduced dataset if the top 40% of peaks have at least an 80% overlap ratio with the compared dataset.

 

High efficiency ChIP on 10,000 cells

ChIP efficiency on 10 000 cells. ChIP was performed on human Hela cells using the Diagenode antibodies H3K4me3 (Cat No. pAb-003-050), H3K27ac (pAb-174-050), H3K9me3 (pAb-056-050) and H3K27me3 (pAb-069-050). Sheared chromatin from 10 000 cells and 0.1 µg (H3K27ac), 0.25 µg (H3K4me3 and H3K27me3) or 0.5 µg (H3K9me3) of the antibody were used per IP. Corresponding amount of IgG was used as control. Quantitative PCR was performed with primers for corresponding positive and negative loci. Figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).