[bruce01]

Hi Susma, that would be very helpful, look forward to your answers.

[susma]

Hi again,

I checked my journal. It was rRNA contamination and I realized that the rRNA depletion kit using was not so efficient. I switched to Ribo-Zero kit and I got nice depletion afterwards and nice libraries.

If you want to make sure that you have the same problem or not, just run your RNA input on bioanalyzer. Then if you see the rRNA peaks (even small), it shows that you have the same problem.

Be careful that you are not overloading RNA when you are using Ribo-Zero kit for depletion. Otherwise, you will see the same spikes in your library traces.

I hope you get over this

Susma

[bruce01]

Many thanks for the information Susma, we were using old Turbo DNAse and RiboZero kits, so this may have resulted in less rRNA depletion, or maybe all DNA was not removed which can result in poor RiboZero performance. Anyway, good to know and we will check on Bioanalyzer for rRNA contamination.

Bruce.

[ruoft]

Just FYI: My bioanalyzer traces looked exactly like this using the same kit. I ran the libraries on a gel and it pretty much replicated the bioanalyzer. Then I took two uL of each library and boiled it in 6uL FA dye to denature the sample and ran in on a 2% TBE gel. The upper bands disappeared and I got a nice smear from ~200-~600 bp. Maybe those spikes are the adaptor annealing products? (I did 14 cycles PCR and started with ~100ng RNA).