Once the initial reverse transcription has occurred, LUTHOR 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, special care should be taken that the cells are viable and stay intact.
Lysis Temperature: Cell suspensions or FACS-sorted cells are lysed during Step 4 of the LUTHOR 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!
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.
At Step 4, ensure that the 4 °C hold is executed and avoid exposing samples to higher temperatures.