Transcriptome Analysis of Tie2Cre;Ino80 fl/fl Mouse Hearts

E13.5 embryos were dissected from Tie2Cre;Ino80 fl/fl pregnant females. RNA from whole hearts were used to prepare sequencing libraries with NEB Next Ultra RNA library prep kit and sequenced with 76 bp Illumina paired-end reads. Raw sequencing library quality was assessed using FastQC. Reads were then mapped to genome GRCm38 (mm10) using STAR (v2.4.2a) with gene annotations from the GENCODE primary assembly (vM6). Reads aligning to transcripts were counted using the summarizeOverlaps function from the GenomicAlignments R package^{54 (link)}. An average of 33.9 M reads were counted for each WT replicate, vs. an average of 53.5 M reads for each KO replicate. Count data were input into DESeq2, and data were regularized-log-transformed prior to visualization by heatmap^{55 (link)}. Significance results and log₂(fold change) values were generated using the DESeq2 algorithm on untransformed count data.
Gene ranks for pre-ranked GSEA were defined as -log₁₀(padj) for upregulated genes in the mutant (log₂(fold change)>0) and (−1) × −log₁₀(padj) for downregulated genes, where padj is the Benjamini-Hochberg corrected p-value from DESeq2. This is analogous to the ranking system used in reference^{56 (link)}. Enrichment of 15597 genes with non-zero expression was evaluated within 7057 hallmark gene sets^{57 (link)} and Zhang et al.^{37 (link)} using the “weighted” enrichment statistic.