How to get rid of Shoulder Peak in Illumina Library

[aspiring]

Hello Everyone!
I am preparing Library using Illumina TruSeq (Starting Total RNA 1.5 microgram with RIN: 10). I have been trying to do this a couple of times but every time I do this a shoulder peak of ~248 bp slowly creeps in. I would be very much thankful if you could please help me explain this and get rid of it.
Thank you very much!

[nucacidhunter]

I am not sure the instrument that you have run the libraries. Those peaks may not be related to library prep. I have seen those when the instrument is not in a stable position for instance the table is moved or it is near a centrifuge and the vibrations affects the instrument during loading.

[luc]

What type of sample are you studying? This might be caused by a predominant short transcript?

[aspiring]

What type of sample are you studying? This might be caused by a predominant short transcript?

Hi Thank you for your response. I am studying HeLa RNA samples. The OD 260/280 and OD 260/230 ratios were 2.03 & 2.01 whereas the RIN value was 10, which shows that the starting RNA sample is not degraded and very pure.

[aspiring]

I am not sure the instrument that you have run the libraries. Those peaks may not be related to library prep. I have seen those when the instrument is not in a stable position for instance the table is moved or it is near a centrifuge and the vibrations affects the instrument during loading.

Thank you for your response. The instrument has always been stable as we have dedicated table for the bio analyzer. There is a small table top centrifuge to spin down the samples but we use it before turning on the bio analyzer.

[nucacidhunter]

It this case luc explanation is more likely the cause. Aligning reads to reference or BLAST search should help to identify overrepresented transcripts/library fragments. Sometimes they could be result of insufficient polyA selection or depletion that can be corrected but sometimes result of biological state of cells which may not require any action.

[aspiring]

I am not sure the instrument that you have run the libraries. Those peaks may not be related to library prep. I have seen those when the instrument is not in a stable position for instance the table is moved or it is near a centrifuge and the vibrations affects the instrument during loading.

It seems as if the stability of tape station affects the generation of peak while running the sample in the tape.