Bovine blastocysts developed with or without telomerase inhibitor TMPyP
Telomere is an important biomarker of embryos which potentially links to the health of offspring This data is RNA-seq of the blastocyst stage embryos having long and short telomere. Ovaries were collected from a slaughterhouse and oocyte-cumulus cells complexes (COCs) were collected from antral follicles of ovaries. COCs were cultured in TCM-199 medium containing 5mM Taurine, 10% FCS and 10ng-mL EGF for 24h. After the maturation, oocytes were subjected to in vitro fertilization, protocol of the fertilization is described in the previous report (Int J Mol Sci. 2023;24:3508. PMID: 36834915). 5, 10, 15, 20-tetrakis (1-methylpyridinium-4-yl) porphyrin p-toluene sulfonate (TMPyP; A5014; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), was added to the IVC medium at a final concentration of 10 ÂµM. Eighteen hours after insemination, presumptive zygotes were incubated in embryo culture medium containing telomerase inhibitor TMPyP. TMPyP is a quadruplex-specific ligand and inhibits telomerase. In this project, blastocysts developed with the inhibitor had significantly shorter telomere comparted with those vehicle (water) counterparts. The RNA extracted from the 35 embryos were subjected to RNA-seq. RNA extraction was conducted using RNAqueous Kit (Invitrogen Waltham, MA, USA). Three samples were prepared from differential ovary series collected from at least 20 cows. The RNA quality and concentration were examined using a Bioanalyzer (Agilent technologies, Palo Alto, CA, USA). Average and standard error mean of RIN was 9.63 Â± 0.11. Libraries were created using the NEBNext Single Cell/Low Input RNA Library Prep Kit (Bio Lab, Ipswich, MA, USA), and library quality and quantity were determined using the Agilent 2100 Bioanalyzer and KAPA Library Quantification Kit (KAPA Biosystems), respectively. The multiplied samples were sequenced as 100 bp reads (single reads) on NextSeq1000 (Illumina, San Diego, USA). Image analysis, base calling, and quality filtering were performed using the RTA version 2.4.11 and bcl2fastq2 v2.20.0.422 (Illumina), following the manufacturer's instructions.