| description |
Fertilization precisely choreographs parental genomes by utilizing gamete-derived cellular factors and by activating genome regulatory programs. Here, we carefully extracted more than 14 x10,000 high-quality mouse metaphase II oocytes with which we established the transcriptional profile of four early-embryo stages using in vitro fertilization and total RNA sequencing. We collected MII oocytes from the oviducts of 8- to 12-week-old superovulated female BBF1 mice, which are F1 progeny from a cross between Balb/c females and C57BL/6 males. After removing cumulus cells, we manually picked high-quality cells (~30 oocytes per mouse). The 1C, 2C, and 4C embryos were obtained by in vitro fertilization followed by addition of nocodazole (0.1 ug/ml in the medium for 12 h) to block cells at prometaphase of the first, second, or third mitosis, respectively. Total RNA (3-4 ug per stage) was prepared from 10,000 cells at each stage using ISOGEN II (NIPPON GENE). To prepare sequence libraries, we first removed rRNA using a Ribo-Zero Gold kit (Epicentre, an Illumina company), which yielded ~500 ng RNA per stage. Then, we randomly fragmented the RNAs using RNase III provided in the SOLiD Total RNA-Seq kit (P/N 4445374). After isolation with gel electrophoresis, we converted 100- to 200-bp RNA fragments to cDNA libraries in a strand-specific manner. Using the standard protocols from the SOLiD System User Manual, the cDNA libraries were clonally amplified onto beads with emulsion PCR. We sequenced the library fragments on a SOLiD 5500xl System Analyzer using standard run conditions and generated 50-bp single-ended short reads. |