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The ability to produce libraries starting with low input DNA is primordial when using ChIP-seq. I am a ChIP-seq user and I always use the MicroPlex kit for library making from my immunoprecipitated DNA. Not only it's quick and requires as little as 50 pg of input DNA but it also never fails. I've made over 100 libraries using the MicroPlex library preparation kit, and none of them failed. I use the Microplex kit for preparing libraries from immunoprecipitated DNA and from genomic DNA with low concentration.Zineb Rchiad, Microbial Genomics Laboratory, King Abdullah University of Science and Technology, Kingdom of Saudi Arabia.
As part of a genomics lab, I regularly prepare libraries from a large number of samples. The IP-star® is extremely useful for high throughput library preparation. I have prepared 200 genomic libraries in less than 1 month using the IP-star®, it has helped me economize time and effort and increase reproducibility. I have used the IP-star® for automation of ChIP as well; 16 samples can be processed in one run. In brief, it's a blessing to have this machine if you apply ChIP-seq to a large number of samples.
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.
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.
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.
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).