Table 2 |.
Troubleshooting table
| Step | Problem | Possible reason | Solution |
|---|---|---|---|
| 4 | Low expression level of exogenous RNASEH1 | Poor effect of hygromycin B | Always include a well of untransfected cells as a control for selection. At an effective concentration of hygromycin B, untransfected cells will be killed witdin 5 d |
| 27 | Low chromatin-shearing quality | Inefficient cell lysis | Incubate the cells in nuclear lysis buffer for a longer time and avoid the formation of cell clumps |
| Insufficient sonication | Increase the cycle number of sonication and always check the sonication efficiency before IP. We also recommend optimization of the sonication conditions for each new experimental setting (different number of cells or different volume of nuclear lysate) or when working with a new cell type | ||
| 43 | Low IP quality | Poor antibody quality | Increase the amount of antibody per sample or switch to another ChIP-grade antibody if the current one has a specificity issue |
| Excessive washing of the chromatin–bead complex | Reduce the incubation time of the chromatin–bead complex with wash buffer II | ||
| 60 | Low R-ChIP enrichment as assessed by qPCR(≤2-fold enrichment) | Low cell quality | Harvest the cells when they are in the active proliferation state and have reached 70–80% confluence |
| Insufficient cross-linking | Use fresh formaldehyde and increase the cross-linking time, but do not use a formaldehyde concentration higher than 1% (vol/vol) | ||
| Poor sonication quality | Optimize the sonication conditions. Avoid overheating of chromatin fractions during sonication | ||
| Insufficient washing of beads | Increase the wash time with wash buffers, especially high- salt wash buffer II | ||
| 85 | Low library yield | Insufficient cell number for the experiment | Prepare more cells before harvesting |
| Sample lost through library construction | Before using IPed DNA, perform Steps 61–85 with an aliquot of input DNA to test the recovery efficiency of the PureLink PCR Micro Kit used, by measuring the concentration of recovered DNA and adaptor ligation efficiency by estimating the PCR product amount by gel electrophoresis | ||
| Poor adaptor quality and ligation efficiency | Check the adaptor purity by size on a PAGE gel after annealing and increase the adaptor concentration during ligation | ||
| Insufficient PCR cycle number | Always run a test PCR to determine the most appropriate cycle number |