I am planning to do single-nuclei sequencing for brain organoids. However, the organoids experienced 25 minutes in room temperature prior to freezing. Most of the time they were in their culture medium, but had a wash (0-2 minutes) in room temperature PBS prior to snap freezing in liquid nitrogen vapour. My questions:
1. Would the nuclear RNA be still intact?
2. How much would the exposure to room temperature impact gene expression?
3. What is your experience in filtering out (during analysis) early response transcripts expected to be upregulated due to processing of the samples?