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
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
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