This tool is based on chipd[1] and provides a starting point for a depletion design. It designs probe sequences using settings that have worked well in our hands ( -tM 3 -Lx 60 -s 15 -tN 0.2) For maximum depletion effectiveness we typically supplement with manually designed probes in any regions that cannot be addressed by the tool. We also typically compare transcript levels between an undepleted and depleted library to confirm that only the targeted species are being depleted.

1. Dufour et al., “ChipD: A Web Tool to Design Oligonucleotide Probes for High-Density Tiling Arrays.”

Desalted DNA oligos are typically of sufficient purity for depletion applications. The synthesis scale will depend on the number of oligos in the pool and the number of reactions desired. We recommend preparing an equimolar probe pool where each probe is at a final concentration of 2 µM. Ordering pre-resuspended oligos (10 mM Tris, 0.1 mM EDTA pH 7.5) is preferable as it reduces hands on time and lowers the risk of cross-contamination. If lyophilized oligos are ordered, ensure that they are resuspended to the required concentration. Specific depletion targets can be added to an existing NEBNext Depletion Kit. Please consult the manual for detailed recommendations or contact technical support for assistance.