RNA-seq analysis of Alyref and Gabpb1 knockout embryos

We have identified Alyref and Gabpb1 as developmentally important genes by siRNA screening. Gene knockout (KO) of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. To gain mechanistic insight into the developmental role of Alyref and Gabpb1, we performed RNA sequencing (RNA-seq) analysis of the KO embryos. Sperm and oocytes were collected from hetero mice (Alyref+/- or Gabpb1+/-), and in vitro fertilization (IVF) was performed ([Alyref+/- x Alyref+/-] or [Gabpb1+/- x Gabpb1+/-]). As a control, IVF using wild type (WT) mice were also carried out. The resulting IVF embryos were cultured in potassium simplex optimized medium (KSOM) at 37°C under 5% CO2. At 96 hours post insemination (hpi), embryos at the morula and blastocyst stages were collected and subjected to single embryo RNA-seq. Single embryo RNA-seq enabled us to distinguish KO embryos (Alyref-/- or Gabpb1-/-) from hetero and WT embryos due to the lack of sequence reads at the deleted region. Then, transcriptomes of KO and other embryos were compared.