Spike-in Control Nuclei Services Overview
Identifying differences between epigenetic assay data sets can be challenging when global modification changes occur, such as when studying the effects of chromatin modifying enzyme inhibitors. Additionally, inaccurate quantification of starting material or technical variation during processing results in variation across sample data. Spike-In controls for ATAC-Seq, CUT&RUN, and CUT&Tag can be invaluable tools that allow researchers to see, compare, and report biological differences between samples with confidence. Active Motif’s Spike-in Control Nuclei is a reliable strategy for sample normalization in ATAC-Seq, CUT&Tag-IT®, CUT&Tag-IT® R-loop, and ChIC/CUT&RUN Services.
For more details on the spike-in strategy check out our Spike-In Normalization Resource Center
Spike-In Control Nuclei Highlights:
- Compare sample datasets
- Reveal global changes in chromatin
- Correct for differences in sample input that could mask biology
Spike-in Control for ATAC-Seq Services
With the Spike-in Control Nuclei add-on option for ATAC-Seq Services, Active Motif will spike in cryopreserved Drosophila cell nuclei into samples prior to tagmentation. During tagmentation both the sample nuclei and Drosophila nuclei are tagged at open chromatin consistently across all samples. A normalization factor is then created based on the Drosophila signal and applied to the test genome.
Spike-in Control for CUT&Tag-IT®, CUT&Tag-IT® R-loop, and ChIC/CUT&RUN Services
For CUT&Tag-IT®, CUT&Tag-IT® R-loop, and ChIC/CUT&RUN services, cryopreserved Drosophila cell nuclei are spiked into samples prior to running the assay. Drosophila H2Av antibody is added concurrently to the target antibody of interest during the primary antibody incubation step. This Drosophila H2Av antibody does not cross react to other species and provides a mechanism to reliably tag Drosophila chromatin consistently across all samples. A normalization factor is then created based on the Drosophila signal and applied to the test genome. This Spike-In Control strategy enables normalization of sample data independent of the experimental antibody and without bias.
Spike-in Control Nuclei Services Data
ATAC-Seq Spike-In Control
Figure 1: K562 Starting Cell Numbers for ATAC-Seq
This experiment used 10,000 drosophila nuclei as a spike in for a mock treatment. The mock treatment was a cell titration performed in duplicate from 200,000 cryopreserved K562 cells down to 10,000 cryopreserved K562 cells. The actual cell numbers are 200,000, 100,000, 50,000, 20,000 and 10,000 cells. This figure is all of the normalized tracks for the titration in duplicates.
CUT&Tag-IT® Spike-In Control
Figure 2: K562 CUT&Tag-IT Assay Results With and Without CUT&Tag-IT Spike-In Control Normalization
IGV browswer tracks are shown for duplicate reactions of 50,000, 100,000, 150,000, and 200,000 K562 cells assayed in CUT&Tag for H3K27me3 without spike-in and with CUT&Tag-IT Spike-In Control normalization. In the Spike-in Normalized panel it is clear that the peak height correlates with increasing cell number, whereas in the No Spike-in control peak height differences are not noticeable from 50,000 to 200,000 cells.
Figure 3: EZH2 inhibition K562 CUT&Tag-IT Assay Results With and Without CUT&Tag-IT Spike-In Control Normalization
IGV browser tracks are shown for control samples and samples treated with an EZH2 inhibitor. Data was analyzed both without and with Spike-in normalization. Global decrease in H3K27me3 histone modification are only detectable with Spike-in normalization.
CUT&RUN Spike-In Control
Figure 4: K562 Paired Alignments with and without Norimalization
The number of alignments is not reflective of the number of cells added to the experiments targeting H3K4me3 (left panel) and YY1 (right panel), thus normalization is needed.
Figure 5: K562 Paired Alignments After Normalization to Drosophila Paired Alignments
K562 paired alignments were normalized to the Drosophila alignment number from 500,000 K562 cells. Now the expected ratios based on amount of starting cells has been restored in H3K4me3 (left panel) and YY1 (right panel).
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Name | Cat No. | Price | |
---|---|---|---|
Spike-in Nuclei Add-on | 25232 | Get Quote |