Hello,
I would like to ask for the opinion of those having experience in this procedure.
We are dealing with bacterial transcriptomics and applying RNA-Seq.
We are following the TruSeq RNA Sample preparation guidelines from Illumina, the Low Sample (LS) protocol, and starting from 0.5 ug of total RNA samples.
Ribo-Zero Magnetic Gold kit was used as starting point to deplete samples from ribosomal RNA.
Ok, so the first issue:
1) the quantification by Qubit of the final libraries gave very low values, ranging form 1.22-7.3
and the second:
2) there was a huge contamination of what seems to be primer-dimer (see attachment)
I am struggling trying to understand where these problems might come and how to solve them in the next preparation, as it is clear that the present libraries cannot go into the sequencer.
Thanks in advance for your comments
Cristina
I would like to ask for the opinion of those having experience in this procedure.
We are dealing with bacterial transcriptomics and applying RNA-Seq.
We are following the TruSeq RNA Sample preparation guidelines from Illumina, the Low Sample (LS) protocol, and starting from 0.5 ug of total RNA samples.
Ribo-Zero Magnetic Gold kit was used as starting point to deplete samples from ribosomal RNA.
Ok, so the first issue:
1) the quantification by Qubit of the final libraries gave very low values, ranging form 1.22-7.3
and the second:
2) there was a huge contamination of what seems to be primer-dimer (see attachment)
I am struggling trying to understand where these problems might come and how to solve them in the next preparation, as it is clear that the present libraries cannot go into the sequencer.
Thanks in advance for your comments
Cristina
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