Evaluation of transcriptomic changes in a Neuro2a cell line under extreme stress

Bulk poly(A) RNA-sequencing was performed to evaluate the transcriptomic profiles of Neuro2a cells exposed to extreme stress following thawing in liquid N2 storage. This RNA-seq experiment was conducted as a part of broader aim in our study to evaluate changes gene expression of stressed cells in the early stages of recovery at Passage 1 post-thaw (P1) and as cells progressed towards a healthy phenotype at Passage 6 post-thaw (P6). Briefly, RNA was extracted and purified from Neuro2a cells at P1 (n=4 for experimental; n=3 for controls) and at P6 (n=3/group); poly(A) RNA was isolated, fragmented, and converted into cDNA libraries. Libraries were sequenced on an Illumina NovaSeq 6000 instrument at an approximate depth of 40 million 150 bp paired-end reads per sample. Sequencing yielded an average of 38.99 million reads per sample with and average of 94.91% of reads having a quality score of at least 30 (Q30) and an average of 96.29% of reads mapping to the mouse reference genome. Overall design: To evaluate the effects of cryostorage failure on cells, we developed a standardized model to recapitulate a liquid nitrogen storage depletion event and subsequent thawing of Neuro2a cells to temperatures of 4-10°C for approximately 24h and subsequently re-freezing and plating Neuro2a cells using a standard protocol. Gene expression was evaluated using RNA-seq at two timepoints: one at the early stage of recovery shortly after stressed cells regained rapid proliferative capacity (Passage 1) and one at a later stage of recovery when cells appeared phenotypically normal (Passage 6). Cells were collected alongside time-matched controls at each passage for poly(A) RNA-seq and comparative gene expression analysis was performed to identify differentially expressed genes.