RNA-seq Analysis of Blastocyst Transcriptomes

Single blastocysts from WT and KO group were collected on day 7. The zona pellucida of blastocysts was discarded with 0.5% pronase E. The RNA-seq libraries were constructed according to Smart-seq2 procedure as previously described (Picelli et al. 2014 (link)). In brief, polyadenylated RNAs were captured and reverse transcribed with an Oligo(dT) primer, then the cDNA was pre-amplified using KAPA HiFi HotStart ReadyMix (kk2601). Pre-amplified cDNA was purified with Ampure XP beads (1:1 ratio) and fragmented by Tn5 enzyme (Vazyme, TD502). PCR amplification for 15–18 cycles was performed to prepare sequencing libraries, which were subject to paired-end 150 bp sequencing on a NovaSeq (Illumina) platform by Novogene. The raw sequencing reads were trimmed with Trimmomatic (version 0.39) (Bolger et al. 2014 (link)) to generate clean data and mapped to ARS-UCD1.2 with Hisat2 (version 2.1.0) (Kim et al. 2015 (link)). The raw counts were calculated with featureCounts (version 1.6.3) (Liao et al. 2014 (link)) and underwent differential expression analysis using DESeq2 (Love et al. 2014 (link)). The differentially expressed WT and KO gene groups were identified using an false discovery rate (FDR)-adjusted P-value (P_adj) less than 0.05. Foldchange ≥1.5 or ≤0.6. Fragments per kilobase million (FPKM) for each sample was calculated with Cufflinks (Trapnell et al. 2012 (link)) for heatmap visualization, and heatmaps were generated using a pheatmap package in R. Gene ontology analysis was performed with the Database for Annotation, Visualization and Integrated Discovery (Huang da et al. 2009a,b (link, link)). All RNA-seq files are available from the Gene Expression Omnibus database (accession number GSE216123).