Clontech Stranded - Multiple Peaks

[ChP29]

Hi everybody,
I tried for the first time the Clontech Stranded mRNA-Seq Kit for Illumina to generate libraries. My starting material (1 ng total RNA isolated from human serum) was ok. However, after library preparation I ended up with a weird looking electropherogram on the Bioanalyzer. My first thought was that this might be due to overamplification, so I reduced the final amplification cycles (12-14-16). Unfortunately, this didn’t solve the problem.
Do anyone has an idea what’s with these libraries with the expected peak at 300 and the additional peaks at ~400 and 600?

PS. Fragmentation time was 2 minutes (I also tried 4 minutes)



Thank you.

[nucacidhunter]

I wonder what is the exact name of kit or can you provide a link to the product web page.

[ChP29]

Hi nucacidhunter,

the Kit I used is the SMARTer® Stranded RNA-Seq Kit (applicable to 100 pg to 100 ng of input RNA)

http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/Next_Gen_Sequencing_Kits/Total_RNA-Seq/Strand-Specific_RNA_Seq_Illumina

[nucacidhunter]

Your post indicates that poly A RNA has been used for input. I wonder what product was used for poly A isolation.

[ChP29]

It's total RNA. Since I used RNA isolated from serum it corresponds more to rRNA depleted RNA (as checked by Bioanalyzer)

[nucacidhunter]

I looked at the protocol and as you mention it does not seem to be a PCR artefact. I have used the recommended low input library prep kit with DNA and PCR fragment size is proportional to input DNA. It could be:
1- Fragmentation issue where some cDNA fragments did not shear well (protocol recommends 5 min) so you have a population of cDNA in various fragments hence more than one peak.
2- The peaks at 400 and 500 bp could be from a particular transcript that over-represented in input RNA. They can prime preferentially at the same region during 1st strand synthesis and result in small dscDNA. Smaller fragments are less likely to be fragmented with sonication and will form their own peak.
3- If you have some poly A RNA (the source does not matter) you can try prepping library using the same amount and increase shearing time to rule out number 1.
4- You can also check purified and sheared cDNA on a Chip to look for size distribution if any has left.

[nucacidhunter]

Other possibility is cell free DNA contamination that has been converted to library. Posting input RNA Bioanalyzer profile would be helpful. If your input RNA fragments were short then you might have used non-optimal kit.

[SS00]

Did you do any pre-processing to the RNA like ribo-depletion? I have seen this with my clontech library prep when I used ribo-zero on my RNA before I put it into Clontech's protocol. It seemed that some salt or other contamination from ribo-zero was interfering with my fragmentation.