Hi,
I have a relatively basic question. I am performing RNA-seq on some small tissue samples and I have constructed my first set of libraries for Illumina sequencing using the Ovation/NuGen SPIA system. My libraries are at about 2ng/ul concentration, or around 6nM. My question is: how do you known when to do PCR amplification of your libraries? I think that my RNA is concentrated enough to cluster directly onto a flow cell, but I have been told that amplification is usually necessary for SPIA. On the other hand, I would like to reduce amplification bias as much as possible.
Any recommendations here? Any rough guidelines that you use to determine whether to amplify or not?
Thanks in advance, zrl
I have a relatively basic question. I am performing RNA-seq on some small tissue samples and I have constructed my first set of libraries for Illumina sequencing using the Ovation/NuGen SPIA system. My libraries are at about 2ng/ul concentration, or around 6nM. My question is: how do you known when to do PCR amplification of your libraries? I think that my RNA is concentrated enough to cluster directly onto a flow cell, but I have been told that amplification is usually necessary for SPIA. On the other hand, I would like to reduce amplification bias as much as possible.
Any recommendations here? Any rough guidelines that you use to determine whether to amplify or not?
Thanks in advance, zrl
Comment