Pepli g wta single cell kit

The collection of zygotes was performed as previously described [18 (link)]. CD1 female mice were super-ovulated with 5 IU PMSG (367222, Calbiochem) and 5 IU hCG (230734, Calbiochem) for 46 h and used for breeding. After release from the oviduct ampullae, the zygotes were injected with siRNA oligos (20 μM) using the Femojet microinjection system (Eppendorf) and then cultured in KSOM medium for different amounts of time.
After culturing the zygotes for 24 h (two-cell stage), whole transcriptome amplification was performed using the PEPLI-g WTA single cell kit (Qiagen, 150063). Following cDNA synthesis and amplification, the product was used for qRT-PCR to access the knockdown efficiency and the expression of pluripotency markers.
For whole-mount staining, blastocysts were collected into embryo GPS dishes (LifeGlobal Group), fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, blocked with 3% BSA solution, and blotted with anti-Oct4 (sc-5279; Santa Cruz) and anti-Nanog (Ab80892; Abcam) antibodies.

Pdgfd mutant embryos and neonatal mice were obtained from Pdgfd^+/−  × Pdgfd^+/− breeding. Briefly, 6–8 weeks old female mice were superovulated by intraperitoneal injection of 5 IU pregnant mare’s serum gonadotropin (PMSG; Solarbio, Beijing, China), and after 48 h, followed by the injection of 5 IU human chorionic gonadotrophin (hCG; Millipore, Billerica, MA, USA). The female mice were then caged with male mice at a one-to-one ratio. The presence of a vaginal plug in a female mouse about 12–20 h after hCG injection marks 0.5 days post copulation.
For mouse blastocyst collection, embryos were flushed from oviducts and uteri using M2 media (Sigma-Aldrich, St. Louis, USA) at E3.5. The blastocysts were subjected to whole transcriptome amplification using the PEPLI-g WTA single cell kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The product was 10-fold diluted, and 1 µL of the diluted product was used for qRT-PCR.
For E10.5 or E12.5 embryo collection, the embryos were dissected from uterus, washed with phosphate buffered saline (PBS) and then grinded in RIPA (Solarbio, Beijing, China) or Trizol Reagent (Invitrogen, Waltham, MA, USA) and subjected to Western blot or qRT-PCR.