What are the critical steps in the LUTHOR HD 3’ mRNA-Seq protocol?

Once the initial reverse transcription has occurred, LUTHOR HD is a highly robust protocol with little risk of generating artifacts, such as adapter dimers. Prior to this stage, however, there are two phases where careful handling is essential to maximize sample quality for library preparation:

Sample Processing: When using cells as input for LUTHOR HD, special care should be taken that the cells are viable and stay intact. We recommend to work with cell viability >90% for best results.

Lysis Temperature: Cell suspensions or FACS-sorted cells are lysed during Step 4 of the LUTHOR HD protocol. Exposure to temperatures > 4 °C at this point may lead to RNA degradation and cause a reduction in the overall quality of the sequencing data, e.g., RNA degradation can lead to an increase in intronic and rRNA reads.

TIPS!

• Low binding tips, tubes, and plates should always be utilized to ensure the sample doesn’t adhere to the plasticware. This is critical when working with ultra-low inputs and single cells.

• For best practice, cells should be kept at 4 °C prior to lysis. Dead or dying cells will result in a decrease in the data quality and may lead to an increase of reads obtained for mitochondrial transcripts.

• Step 5: ERE solution to be diluted 1:10 shortly before using (minimum of 8 reactions per experiment).

• Step 7: Process to purification immediately after THOR reaction.

• Step 13: Proceed to purification immediately after IVT or incubate IVT overnight at 4 °C (or stop and store at -20 °C).