Is CORALL Total RNA-Seq V2 suitable for use with degraded or FFPE RNA samples?
Yes, low quality and FFPE samples can be used with CORALL Total RNA-Seq, where a prior ribosomal RNA depletion step is included. However, RTL is not recommended for use with degraded / FFPE RNA samples. For degraded / FFPE RNA samples, please use RTM.
Protocol modifications are recommended for library preparation from degraded RNA and FFPE samples when using the CORALL Library Prep Kit.
The following recommendations have been evaluated with 25 – 500 ng total RNA input:
Perform DNase I treatment prior to rRNA depletion, without an intermediate cleanup step.
Perform ribosomal RNA depletion (e.g., using Lexogen's RiboCop rRNA Depletion Kits).
In step 33 of the CORALL V2 Library Prep Protocol, use 42 µl of PB (instead of 31.5 µl).
When using Lexogen's Spike-In RNA Variant controls (SIRVs), these should be spiked into the Total RNA prior to DNase I treatment, The recommended spike-in amount should be reduced to target 0.1 – 0.2 % of sequencing reads that map to SIRVs (FSIRV ; Explained further in the SIRV User Guides and our How to use SIRVs FAQs. SIRV spike-in calculation worksheets are available from the SIRVs Download Page.
A minimum sequencing read length of 75 bp is recommended. Single or Paired-End sequencing formats can be used.
For further questions, please contact technical support.