I am considering using Illumina for RNA-seq to measure gene expression in a prokaryotic genome of ~ 6Mb. Cost is an issue, but I am trying to make sure I take into account biological and technical replicates, as I have seen many comments about the importance of replicates, etc.
My experiment will yield 8 prokaryotic samples with 3 biological replicates for each sample. I am thinking that to save money I can prepare the libraries myself using the TruSeq kit and order extra barcodes so each biological replicate would have its own barcode, for a total of 24 barcodes that I could sequence in one lane. Then I would perform my technical repeat in a second lane of sequencing on the same libraries. My question is whether or not that would that be a sound technical repeat? Can I use the same libraries between technical repeats? Also, any recommendations on how many reads per sample? I just saw a paper saying 500 million or more for eukaryotic, but what about prokaryotic? Also, is there any value to paired-end reads versus single-end reads for prokaryotic RNA-seq?
I am new to NGS, and I must apologize for how basic some of my questions may be--any advice is most welcome!
My experiment will yield 8 prokaryotic samples with 3 biological replicates for each sample. I am thinking that to save money I can prepare the libraries myself using the TruSeq kit and order extra barcodes so each biological replicate would have its own barcode, for a total of 24 barcodes that I could sequence in one lane. Then I would perform my technical repeat in a second lane of sequencing on the same libraries. My question is whether or not that would that be a sound technical repeat? Can I use the same libraries between technical repeats? Also, any recommendations on how many reads per sample? I just saw a paper saying 500 million or more for eukaryotic, but what about prokaryotic? Also, is there any value to paired-end reads versus single-end reads for prokaryotic RNA-seq?
I am new to NGS, and I must apologize for how basic some of my questions may be--any advice is most welcome!
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