Can you post an image of the Bioanalyzer?

Otherwise, I agree with Mathew that trailing peaks are usually not a problem.

Thanks Mathew. I believe too that perhaps the trailing peak is due to either overamplification since I started with 4 ug of RNA or due to skewed shearing. 

I do purify the cDNA with AMPure XP beads after each reaction step and that serves to generate the pure cDNA. However I do not use SPRI selection. Is it recommended to work with the TruSeq LT stranded mRNA Prep Kit? Perhaps you could throw some light on this matter??

Hi Emanuel. The Bio-analyzer I use is the Agilent 2100 instrument. I have attached an online image representative of the actual instrument that I use at my Institute.

Thanks so much Mathew for the insightful reply. Indeed the inclusion of the SPRI size selection step seems to be a viable inclusion into the current protocol.

However as for the TruSeq mRNA LT kit, the recommended range for preparation of the cDNA was given to be 0.5 - 4 ug. I probably used the maximum recommended amount since this was my first time with RNA-Seq prep. However, some sequencing experts are saying that in all probability the bit of tailing that I see at the end of my library might be due to over-amplification. I don't yet know though if it will affect the quality of the sequencing adversely though.

Hi everybody, sorry for my delay, and when asking for the Bioanalyzer image I actually meant the trace – I have seen only now that you have already posted the PDF in the first question.

I agree with Matthew that the library should be of sufficient quality for sequencing, and that 4µg is quite a lot. We usually use 1 µg of high-quality RNA (i.e. directly extracted from cells/tissue). Furthermore, we have recently begun to use only 60% of a reaction to save money, and can still amplify with only 8 PCR cycles.

Tushar, how many PCR cycles did you apply?

To end with the SPRI – Ampure XP are actually SPRI beads, so there are lots of size selections within the Truseq protocols, which is based on the ratio of beads to reaction volume. You might have noticed, that this ratio is different after the ligation step (in order to remove adapter dimers). See also http://core-genomics.blogspot.de/2012/04/how-do-spri-beads-work.html

I used 13 cycles of PCR for the final amplification, which is what I feel may have led to my trailing peaks. Seeing the Bioanalyzer traces from my data, it seems like if and when I use 4 ug of RNA then around maybe 11 cycles would be more than sufficient. Or perhaps even lesser for that matter.

I will try this the next time around when I prepare samples.

Thanks guys. :) I have learnt quite a bit from you both in terms of RNA-Seq libraries!!

13 cycles is quite a lot, especially with 4 µg starting material. We routinely do qPCR to determine cycle numbers: 10% of the reaction before PCR, with some of the PCR primer mix, and 2x SYBR green qPCR master mix. We then take a cycle number that is somewhere in the second half of the exponential phase of the qPCR curve.