Statistical analysis
Ratios extracted from Proteome discoverer were log2 transformed and
quantiles normalized in R. This data was then used to perform an
empirical Bayes moderated t -test using the limma package
in R (Phipson et al., 2016; Ritchie et al., 2015). Effect of the
parameter day, BSO treatment, and process have been included in the
design matrix. Differentially expressed proteins were identified using
an adj. p. value <0.05. To narrow down the analysis, we
focused only on proteins differentially expressed because of the BSO
treatment and with a log fold change (logFC) threshold of 0.5. A heatmap
of the differentially expressed proteins was generated by hierarchical
clustering using pheatmap package in R (Kolde, 2015). Functional
analyses were performed using MetaCore (Clarivate Analytics, version
19.2.69700) after protein ID conversion to the Mus musculusequivalents. The ID conversion was performed using InParanoid 8 and
UniProtKB BLAST when no matches were found in the first method
(Sonnhammer et al., 2015).