Atto-Seq™, SeqLL’s RNA Seq Sample Prep
The sample preparation steps involved with RNA Sequencing using SeqLL technology starts with taking total RNA (with DRS – Direct RNA sequencing, we can start with less sample material because there is no Ribosomal depletion step.) which is processed as follows:
- Treatmenwith DNAse - an endonuclease which degrades the DNA through cleaving the unwanted portions, releasing the 3’ and 5’ ends, and eliminating genomic contamination.
- The RNA is then put through an rRNA depletion step wherein the Ribosomal RNA, which comprises 95% of the original sample material, is removed.
- The remaining RNA (including Non Coding RNA and mRNA….) is reverse transcribed into single stranded cDNA.
- The cDNA has a Poly-A Tail added to its 3’ end, is blocked with ddATP and is now ready to be hybridized to the oligo dT primed flow cell.
- Once on the flow cell, in order to ensure that sequencing proceeds in the correction direction and that it starts at the correct location – meaning after the Oligo dT and Oligo As which were added to adhere the sample to the flow cell – a fill and lock procedure occurs by adding dTTP to the remaining A tail and a virtual terminator nucleotide (labeled with a fluorescent die) is inserted at the first nucleotide that is not an A.
If rRNA is not problematic, then the rRNA depletion step can be skipped and the amount of sample needed to start will be greatly reduced (to between 50 to 200ng) for the cDNA synthesis protocol.
To learn more about the SeqLL’s RNA sequencing sample preparation contact us!
