Epicentre kit & Bioanalyzer result

[wacguy]

Hi there,

I start constructing RNA-seq libraries after a few month of planning it, from Arabidopsis root material. My initial sample size is very low but using RNAzol, I can get total of 10-15ng high quoality RNA in 10microliter. I decided to try and make a stranded library using the Epicemtre Ribo-zero (for leaf, it is their best kit now and was recommended by their rep) followed by the ScriptSeq v2 RNA-Seq Library Preparation Kit (although the RZ requires 100ng and I had ~10). I was following the protocol (including the AmpureXP) and made 18 PCR cycle as a last step (using index # 1 reverse primer) and surprisingly got a nice library. It looks good on a gel, could amplify 3 genes by PCR but I want some opinions about the bioanalyzer (attached) result: first curve is my library and the second is a colleague + control. Specifically, what are 2 picks around 46 bp (primer dimer) and why do I have the shoulder of long fragments. Last, the index primers kit mentions that each primer (reverse) is 64 bases, seems very long to me.

Thanks a lot,
Guy

[CHHALD]

Dear Guy

I can see from your post that you tried the Scriptseq v2 kit using 10 ng total RNA as input. I have the same problem as you with low levels of starting material, although I have about 30 ng per sample.

I am hoping that you could let me know if you ended up using the Scriptseq v2 kit, and if you were happy with your results.

Best,
Christa

[wacguy]

Hi Christa,

Yes, I was using it and it all works well (for the Ribo-zero, I use half of the amounts as one of their reps suggested me, and it also saves money). I succefuly ran the Toxedo package and could identify many interesting things, the only problem is that I had very low mapability, which I still don't know the reason for.

Let me know if uoi have any more questions,
Guy

[CHHALD]

Dear Guy

Thank you for the quick answer! I will go on to try it myself!

Best,
Christa