Hi everyone, I'm supposed to start some sequencing for small RNAs using the MiSeq and I have some questions regarding the library prep. It's more of a theoretical question rather than a protocol-based one and due to my inexperience with sequencing, I'm hoping someone can sort of walk me through this (so please be gentle, I feel really dumb for asking this).
I'll be using the NEBNext Small RNA Library Prep Set for Illumina and in the last step of library prep before size selection, we are to perform PCR amplification of the cDNA we have just reverse transcribed in the step before. Here we add small RNA sequencing primers for Illumina as well as index primers as components for the PCR reaction. I understand that index primers are required to do multiplexing in the sequencing step (?) but what's the role of the small RNA sequencing primers? Do they play a role in the library clonal amplification that takes place during sequencing?
It just seems that apart from the adaptors there seem to be a whole lot of other stuff that ends up in the final PCR product that goes into the sequencing so I'm just trying to clarify what's doing what.