For the first microarray analysis, ICM-outgrowths were collected on days-3, -5, -7, and -9 from seeded E3.5 blastocysts as well as from isolated ICMs and ES cells, in three biological replicates. Between 20 to 30 ICM-outgrowths were picked and pooled for each biological replicate. For the second microarray analysis, we collected new samples in a time resolution experiment that included ICM outgrowths on days-0.5, -1, -2, -3, -5 and ES cells that were in passages -2, -4, and-15. In addition, we collected ICM-outgrowths cultivated in N2B27 supplemented with SB431542 on days -1 (D1-SB) and -3 (D3-SB) as the negative control. Between 30 to 40 ICM-outgrowths were picked and pooled in two biological replicates.
Growth protocol
For ICM isolation, the E3.5 blastocysts were submitted to zona removal using acidic Tyrode’s solution (pH=2.2), then placed in a mouse trophoblast antibody for 40 min. Next, blastocysts were treated by guinea pig complement in 50 μl droplets under oil for 10 min. After bubbling and lysis of the trophectoderm cells, they were removed by pipetting. The isolated ICMs were washed in PBS twice before they were picked for microarray analysis. Mouse ES cells were derived by transferring the zona-free E3.5 blastocyst/isolated ICM on gelatin coated plates (0.1%, Sigma-Aldrich) that contained N2B27 defined medium supplemented with R2i (which included 1µM PD0325901 (Stemgent) and 10µM SB431542 (Sigma-Aldrich)) as well as 1000 U/ml LIF (ESGRO, Millipore). The time when the E3.5 blastocysts/isolated ICMs were transferred into ES cell culture was designated as day 0.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the AllPrep DNA/RNA Micro Kit (Qiagen). Integrity and quality of RNA samples was checked using a RNA 2100 Bioanalyzer (Agilent).
Label
Biotin
Label protocol
RNA samples (RIN > 9) were subjected to a two-round amplification performed using the TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0 (Epicentre) according to the manufacturer’s instructions.
Hybridization protocol
Purified and labeled cRNA was used for each hybridization reaction onto BeadChip Array Mouse WG-6 and MouseRef-8 v2.0 (Illumina, San Diego, CA, USA).
Scan protocol
Scanning was performed using the iScan reader (Illumina, San Diego, CA, USA). The raw signal intensities of regular and control probes were extracted from Illumina® GenomeStudio software
Description
SAMPLE 2
Data processing
RAW expression values were extracted from Illumina GenomeStudio, and were background corrected and quantile normalized by “neqc” function of the R/Bioconductor package “limma” [PMID: 25605792]. Differentially expressed genes were identified by the Empirical Bayesian method of the package limma. The genes with a minimum absolute log2 fold-change of 1 and Benjamini-Hochberg adjusted p-value of 0.01 were considered significant. Subsequent analysis was performed using custom scripts in R programming language
