Femtojet microinjector

All small interfering RNAs (siRNAs) were purchased from GenePharma. Ago2-siRNAs was designed to specifically target Ago2 and on the basis of 30%-52% GC content and avoiding of internal repeats (5′–3′). Ago2-siRNA1: GCAAAGAUCGCAUCUUUAATT; Ago2-siRNA2: GCCAGUGAUCGAGUUUGUUTT; Ago2-siRNA3: GCAGAAACACACCUACCUUTT; the scrambled siRNA used as negative control. All siRNAs were modified with 2′Fluoro rU/C to increase their annealing temperature. The prediction of off-target effects was described in result. To perform microinjection, zygote-stage embryos were placed in 150 µg/mL hyaluronidase (Sigma) to digest the outer granule cells. SiRNAs was centrifuged at 12,000 rpm for 10 minutes at 4 °C and placed at 4 °C for use. Then, siRNA microinjection was carried out with an Eppendorf FemtoJet microinjector and Narishige NT-88NE micromanipulators. For injection, a glass capillary Femtotip II (Eppendorf) was loaded with 2 μL of 10 µM siRNA by a microloader (Eppendorf), and the solution was injected into the cytoplasm in a 100 μL drop of M2 medium (Sigma) plus 5 μg/mL cytochalasin B (Sigma). The injection volume was approximately 2–5 pL. The injection conditions consisted of 250 hPa injection pressure, 60 hPa compensation pressure and 0.7 s injection time. Immediately after microinjection, embryos were cultured in KSOM medium at 37 °C in 5% CO₂.

The Lin28a pooled siRNA and non-targeting pooled siRNA (Dharmacon Germany) were resuspended in RNase free water according to the manufacturer’s instructions and stored in single-use aliquots at −80 °C. siRNAs were microinjected using an Eppendorf FemtoJet microinjector and Narishige micromanipulators. Microinjection pipettes were pulled with a Sutter P-97 pipette puller. siRNA solution (2 μL of 50 μmol/L) was loaded into the pipette and ~5 pL was injected into the cytoplasm of each zygote. A relatively consistent amount was carefully injected each time. After injection, zygotes were rinsed and cultured at 37 °C with 5% CO₂. The sequences of siRNA used are listed in Table S3.