A mixed model with conditions (stirring/shearing versus unstirred/unsheared, or 5 min versus 120 min, respectively) and mouse strain (WT versus Thbs1−/−) as fixed factors and date of experiments and animal within date of experiment as nested random factors were used for ANOVA.
Mice were handled prior to the date of experiments to minimize stress.
Normalised gene expression values (FPKM) were computed with CuffDiff and corrected for GC content, ratio of average gene body coverage at 5′/3′ ends of the gene, using linear models and for date of experiment and date of library preparation using ComBat70.
After birth all the litters were housed with their mother until the date of experiment.
To ensure the homogeneity of collected data across several litters and generations of congenic and control rats, a GLM testing for various factors (such as seasonal variation, litter size, date of birth, date of experiment) was performed for each phenotype.
Comparisons between WT and Thbs1−/− mice for individual time points across experimental conditions (date of experiment) by means of Student's t-test for independent samples were also included, with the recognition that this analysis does not reflect baseline conditions and environmental confounders.
Plates were designated to indicate its contents and date of experiment.
Fixed effects included Food Concentration, Salinity, Population, Block (date of experiment), Food × Salinity, Population × Food Concentration, Population × Salinity, and Population × Food Concentration × Salinity.
SD: standard deviation; IQR: inter-quartile range; * Until date of experiment; † Between collection and centrifugation; ‡ Between centrifugation and storage at liquid nitrogen; § Independent samples T test; # Mann-Whitney U test.
Sowing dates of LD experiments were February 20 , 2009(LD09 code), March 15, 2010 (LD10 code), and April 27, 2011 (LD11 code), while sowing dates of SD experiments were August 15, 2009 (SD09 code) and September 21, 2010 (SD10 code).
