Please note that the following RNA-seq recommendations are necessarily very generic and simplified. They do apply to common scenarios but might not be right for your project. We always suggest a (free) consultation with our staff and with the Bioinformatics Core before the starting a project.
- We like to receive total RNA samples (please see sample requirements in the RNA-seq libraries section: https://dnatech.genomecenter.ucdavis.edu/illumina-library-construction/ ).
- Total RNA samples are required because they do allow to QC the integrity of the mRNA using the rRNA peaks as markers.
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- We do not isolate RNA (nor DNA) in our lab, but the Taqman Core ( https://www.vetmed.ucdavis.edu/vme/taqmanservice/research_svc.html ) can help you with these isolations. If you let them know that the samples are designated for NGS sequencing, the Taqman Core will isolate high-quality total RNA samples by hand and also apply the required DNAse digestion.
- In case you are working with small sample amounts, we would suggest to use RNA isolation kits from Norgen Biotek (https://norgenbiotek.com/category/rna-purification-kits). These kits use a proprietary column matrix (silicon carbide) with a specific very high affinity for RNA (in contrast to the standard silica columns) and thus often provide high total RNA sample yields. In case you are using a Trizol protocol please clean up the samples on a column (like RNA-clean & concentrator) to remove any solvent traces.
- Since you seem to be interested in DGE analysis exclusively, the suggest library prep method would be 3'-Tag-Seq.
- The RNA samples need to be QC-ed on a Bioanalyzer and or the LabChip Gx. I would budget a total of at least $600 for this QC. Each 3'-Tag-Seq library prep would cost $80. For these type of libraries you would require one or two HiSeq 4000 sequencing lanes worth of data ( $1,127 per lane). You could sequence one lane first, analyze the data, and decide then if you require a second lane.