Troubleshooting Guide for use with the NEBNext® RNA Depletion Core Reagent Set and NEBNext Custom RNA Depletion Tool

For help troubleshooting your customized RNA depletion using the NEBNext RNA Depletion Core Reagent Set and NEBNext Custom RNA Depletion Design Tool, refer to the list below to find our suggestions for solving your problem. If you’ve tried these suggestions and are still having difficulty, please reach out to Technical Support.

To return to the NEBNext RNA Depletion Core Reagent Set product pages, click here for NEB #E7865 (without beads) or NEB #E7870 (with beads). The NEBNext Custom RNA Depletion Tool can be accessed here.

Observation Possible Cause Solution

% of reads mapping to the targeted sequence did not decrease after depletion.

Target sequence used as input in the NEBNext Custom RNA Depletion Design tool is not RNA, but cDNA.

Verify your sequence source.

Probes are not covering the area being evaluated for depletion.

Align the probes against the target sequence using your preferred aligner (e.g. Bowtie or bwa) and visualize the probe alignments along with your aligned reads  (e.g. IGV or IGB). Probes should be reverse complement to the target sequences. Look for gaps in the area covered by the probes. Do these gaps correspond to the areas with high read coverage?
If so, design probes against the gap region using the Custom RNA Depletion Tool.
The additional probes can be spiked into the original pool for testing.
We recommend running a no-treatment control for comparison.
Note that small gaps (~15nt) between probes and overlap are expected in the design when using the tool. Such short fragments will be eliminated during library cleanup.

Probe integrity is compromised.

Order the probes from a trusted oligo synthesis provider and store appropriately.
Evaluate your pool using a single-stranded DNA size estimation method to ensure that the length distribution is between 40 and 60 nt.

DNA Contamination of input RNA.

Contaminating DNA can cause inaccurate RNA quantification and impede proper RNA removal.
If the total RNA sample may contain DNA contamination, treat the sample with DNase I (not provided in this kit) to remove all traces of DNA. After treatment with DNase I the enzyme should be removed from the sample. Any residual activity of DNase I will degrade the single stranded DNA probes necessary for the ribosomal depletion.

DNase Contamination of input RNA.

Purify the RNA from DNase I enzyme after treatment. Residual activity of DNase I will degrade the single stranded DNA probes necessary for the ribosomal depletion.

Other Contaminants of input RNA.

Ensure the RNA samples are free of salts (e.g., Mg2+, or guanidinium salts) or organics (e.g., phenol and ethanol), and in nuclease-free water.

Probe hybridization step was not successful.

Ensure that the temperature ramp down during the probe hybridization step occurs at 0.1°C/s. This step should take approximately 20min.

Depletion is not uniform across targeted sequences.

Probe amount needs to be optimized for your sample.

Titrate the amount of probe pool used. If specific targeted regions are not being depleted, increase the relative probe amount of that region in the pool.

Target sequence used to design the probes is different from that used to evaluate depletion.

Genome annotations are constantly evolving. Check for differences between the transcriptome/genome version used to extract the target RNA sequence and that used to evaluate depletion.