How should cells be collected for cDNA synthesis?
How should cells be collected for cDNA synthesis?
For cDNA synthesis directly from cells, it is essential that the cells remain undamaged during cell collection, as damaged cells may contain compromised DNA that can act as a template for reverse transcriptase and contaminate the final cDNA library.
If feasible, we recommend verification of cell integrity prior to FACS collection. Please note that the low-pressure setting should be used on the flow sorting system and that the stream carrying cells should be aimed at the center of the bottom of a prechilled collection well/tube.
We also recommend a preliminary cell sorting pilot study: sort 50, 100, and 1,000 cells using the appropriate nozzle and pressure settings to determine the transfer volume of the sheath fluid. If the actual sheath fluid transfer volume varies from the predicted transfer volume, decrease the volume of nuclease-free water used to prepare the cell collection buffer. See additional information in the ""Supplementary protocol for processing intact-cell inputs with SMARTer Pico v2,"" available on request.