133 A novel liquid nitrogen-free snap freezer Figure 1. Benchmarking performance of snap freezer versus liquid nitrogen quenching. Study design to compare molecular profiles of biospecimen frozen using the snap freezer vs golden standard of liquid nitrogen quenching. A. K562 suspension cell line samples frozen using liquid nitrogen (-196 °C) versus snap freezer (- 73 °C). Positive control samples were kept at room temperature for two hours before freezing in liquid nitrogen. The surplus lysate of each of the nine samples was used for RNA extraction to perform sequencing and determine RNA integrity scores. B. Normal human liver tissue frozen using liquid nitrogen versus snap freezer (2 biological replicates per condition). From each biological replicate, one sample was processed for global (TiOx ) phosphoproteomics and one sample was processed for RNA extraction to perform sequencing and determine RNA integrity scores. INFLUENCE OF FREEZING RATE ON PHOSPHOPROTEOMICS PROFILE Fifteen aliquots of 300 µl HCT116 lysate, each corresponding to 300-400 µg of protein, were placed in three types of vials with different thermal conduction properties (polypropylene, aluminum and aluminum vials covered in paper tape) to influence their freezing rates. For each condition three vials were individually immersed in either LN2 or precooled isopentane for 1 minute and cooled to a temperature of -196 °C or -80 °C, respectively, using a stainless steel vial holder. Pre-cooled isopentane was tested as second coolant, because at room temperature, isopentane (with boiling point of 36.9 °C) is in liquid phase. Therefore, no boiling occurs and the cooling rate is not subjected to the Leidenfrost effect, which is the phenomenon that a vapor layer is formed that prevents heat transfer18,19. The aluminum vial covered in paper tape was not immersed in isopentane, because previously published experiments have shown that this vial was not subjected to the Leidenfrost effect in LN2 20. After adequate cooling, vials were transported using a LN2 container and stored at -80 °C until further use. 5
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