RNA-Seq profiling of Sirt1 deficient mouse oocytes to investigate Sirt1 role in maternal-zygotic transition
RNA-Seq profiling of Sirt1 deficient mouse oocytes to investigate Sirt1 role in maternal-zygotic transition
10-12 weeks old female mice were used as oocyte donors. For the collection of in vivo ovulated (IVO) matured oocytes, PMSG administration was followed by hCG 48 h later; 15–16 h post-hCG injection, ovulated cumulus-oocyte complexes were flushed from the fallopian tube and treated with 0.1 mg/ml hyaluronidase (Sigma-Aldrich, #H3506) in M2 medium for 5 min to remove cumulus cells. Matured IVO oocytes were incubated in potassium simplex optimisation medium (KSOM), modified by 0.1 % of bovine serum albumin (BSA; mKSOM). For the preparation of activating medium, the mKSOM was supplemented with 2 mM EGTA, 10 mM SrCl2, and 5 μg/mL cytochalasin B, for 5.5 h at 37 °C and 5 % CO2. Cycloheximide (CHX; 15 µg/ml) was used for the inhibition of proteosynthesis during the oocyte activation. Afterwards, embryos were moved into pure mKSOM and cultured for 24 to cleaved embryos stages. For each sample, approximately 50 oocytes were collected in TRIzol reagent (Invitrogen, USA) and homogenised, using TissueLyser LT (Qiagen, The Netherlands) for 5 min at 50 Hz. Chloroform was used for phase separation; the aqueous phase was mixed 1:1 with 70 % ethanol and applied to RNeasy MinElute spin columns (Qiagen). Wash and elute steps were done according to the column manufacturer´s instructions. Isolated RNA was processed, using the Takara Smarter Stranded Total RNA-seq Kit v2 Pico Input Mammalian (PN:634413), according to the manufacturer's instructions.