This will be my first time making RNA-seq libraries. I have the RiboZero (for bacterial RNA) and ScriptSeq library prep kit.
I prepared bacterial total RNA and did on-column DNase treatment. NanoDrop 260:280 ratios are 2.0-2.2. The RiboZero protocol recommends quantifying RNA concentrations using Qubit, so I used the RNA Broad Range assay for that. For kicks, I also tried the dsDNA High Sensitivity assay on the same samples.
I am getting about 6-8% genomic DNA contamination compared to the concentration of RNA, but naively, I was expecting much less.
Has anyone done similar DNA vs RNA Qubit measurements for their samples for library prep, and what ranges do you get?
Also, how bad is my 6-8% contamination for RiboZero and subsequent library prep, since the genomic DNA will still be present after rRNA depletion?
Since a RNA purification step is required after RiboZero, perhaps I can proceed with RiboZero first, then do an additional DNase treatment immediately after, and then proceed with the RNA purification step? Or should I not worry about the 6-8% of DNA?
Any recommendations or advice on how to proceed would be much appreciated! I am completely new to this...
I prepared bacterial total RNA and did on-column DNase treatment. NanoDrop 260:280 ratios are 2.0-2.2. The RiboZero protocol recommends quantifying RNA concentrations using Qubit, so I used the RNA Broad Range assay for that. For kicks, I also tried the dsDNA High Sensitivity assay on the same samples.
I am getting about 6-8% genomic DNA contamination compared to the concentration of RNA, but naively, I was expecting much less.
Has anyone done similar DNA vs RNA Qubit measurements for their samples for library prep, and what ranges do you get?
Also, how bad is my 6-8% contamination for RiboZero and subsequent library prep, since the genomic DNA will still be present after rRNA depletion?
Since a RNA purification step is required after RiboZero, perhaps I can proceed with RiboZero first, then do an additional DNase treatment immediately after, and then proceed with the RNA purification step? Or should I not worry about the 6-8% of DNA?
Any recommendations or advice on how to proceed would be much appreciated! I am completely new to this...
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