Contamination with bacterial rRNA

[JakobHedegaard]

We have done Ribo-Zero-Human rRNA depletion of total-RNA from human clinical samples followed by ScriptSeq library prep and HiSeq sequencing. But it turned out that app 50 % of the reads were of bacterial rRNA origin.
Anyone observed something similar?
/Jakob

[Olaf Blue]

Ribo Zero combination H/MR and bacteria

Hello Jakob,

The "fix" actually is simple, and it would involve using the RZ Kits in tandem. After the total RNA depletion, you can use RZ Human with the RZ bacteria (three kinds available - Gram Positive, Gram negative and "meta-Bacterial" for both gram positive and negative) in a single step (mixing the rRNA removal solutions in a 50/50 mix) or running the depletions sequentially.

For the "combined" reaction, after total RNA purification simply use a mix of the RZ Human rRNA removal soln with the bacterial rRNA removal soln of choice. Use 5 µl of the human and 5 µl of the bacterial (total volume of 10 µl for 5 µg input). Them simply continue after the incubation at 50C, and follow the bead-binding and filtration as per normal.

Only caveat here is to not use more than 2.5 µg of total "mixed" sample RNA (due to the overall reduction in the rRNA removal solution from what the standard volume is used in a "normal" depletion.

If you need further clarification, do contact me.

olaf

Epicentre tech support