Command console software 4

RNA extraction and purification, RNA amplification and labeling, array hybridization, and data acquisition were performed as described in the Affymetrix (Santa Clara, CA, USA) manufacturer’s instructions. Affymetrix Human U133 Plus 2.0 whole genome chips were used. Array hybridization and wash was performed using GeneChip Hybridization, Wash and Stain Kit (cat. no. 900720, Affymetrix) in Hybridization Oven 645 (cat. no. 00-0331-220V, Affymetrix) and Fluidics Station 450 (cat. no. 00-0079, Affymetrix) following the manufacturer’s instructions. Slides were scanned by GeneChip Scanner 3000 (cat. no. 00-00212, Affymetrix) and Command Console Software 4.0 (Affymetrix) with default settings. Raw data were normalized by MAS 5.0 algorithm, Gene Spring Software 11.0 (Agilent Technologies, Santa Clara, CA, USA).

Total RNA was isolated from the nasal mucosa samples, three mice each group, using the RNeasy mini kit (Qiagen, Valencia, CA, USA). Total cRNA was generated using a WT Expression Kit (Ambion; Austin, TX, USA) and labeled using a GeneChipWT Terminal Labeling Kit (Affymetrix; Santa Clara, CA, USA). The labeled cRNA was hybridized to GeneChip Mouse Gene 1.0 ST arrays (Affymetrix, Santa Clara, CA, USA) at 45 °C for 16 h and at the end of hybridization, the arrays were washed using the Fluidics station 450, prior to being scanned with a GeneChip Scanner 3000 7G (Affymetrix). The images of all arrays were transformed into digital data using the Command Console Software 4.0 (Affymetrix), and the data for each array were normalized by RMA + DABG normalization using the expression console.

Slides were scanned by GeneChip® Scanner 3,000 (Cat#00–00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 4.0 (Affymetrix, Santa Clara, CA, US) with default settings. Raw data were normalized by MAS 5.0 algorithm, Affymetrix packages in R. Probe sets with signal values lower than the detectable range were adjusted to 75 and probe sets with the values of 75 for all conditions were removed from subsequent analysis. The averages of normalized ratios were calculated by dividing the average of the normalized signal channel intensity by the average of the normalized control channel intensity. The standard deviation of the ground control (two biological replicates) was employed to identify genes of significant changes relative to the ground controls (p value < 0.05). Only genes that showed transcript level changes in at least two folds in comparison with its ground control and with the same tendency in both biological replicates were considered as relevant for spaceflight. Gene Ontology (GO) over representation was performed using PANTHER (Fisher’s exact type with false discovery rate correction, http://www.pantherdb.org, Mi et al., 2019 (link)). For motif enrichment, motifscan (Sun et al., 2018 (link)) was used to determine whether the occurrence of a given motif in input genes was significantly high as compared with that in random regions (Ran et al., 2019 (link)).