GSM8283475: Morula_Ctrl_2; Mus musculus; miRNA-Seq
Znhit3KI/- female mice were injected intraperitoneally with eCG (5 IU) followed 46 h to 48 h with hCG (5 IU) and then co-caged with Znhit3KI/- males. Fertilized zygotes were flushed from plugged females 16 h post hCG injection and cultured in M2 medium containing hyaluronidase to remove the cumulus mass. Zygotes without cumulus were then washed and cultured in advanced KSOM until intended developmental stages. Before sample collection, embryos were first examined by immunofluorescence (IF) staining to confirm expression of the HA tag fused to Znhit3 in the knock-in (KI) allele. All IF reagents were supplied with 0.5U/μl RNase inhibitor. Embryos without the knock-in allele were treated as KO and others as control. 15~20 zona-free KO or control embryos were used as one group which were washed in PBS and then transferred into acidic Tyrode's solution to remove zonae pellucidae before collecting into PCR strips with 5 µl RLT plus lysis buffer. 9 μl RNAclear beads (1:1.8 ratio) were used to purify RNA in each group. 10 μl 50 mM Na2CO3/NaHCO3 buffer (pH 9.2) were used for elution with 5min incubation before heating at 95 °C for 10min in a PCR cycler to finish hydrolysis. 1μl 1N HCl was added to bring down the pH. 22 ul RNAclear XP beads and 28ul 100% isopropanol were then added to bind the fragmented RNA in each group. The beads were washed twice with 180ul 85% ethanol and eluted with 10μl Antarctic phosphatase reaction mix containing 7.5μl H2O, 1μl 10x Antarctic phosphatase reaction buffer, 1μl Antarctic phosphatase (NEB M0289S) and 0.5μl 20U/μl RNase inhibitor. Incubation at 37 °C for 30min and then 70 °C for 5min was performed to finish de-phosphorylation and enzyme inactivation. After de-phosphorylation, RNA fragments were purified again by adding 18ul RNAclear XP beads and 27ul 100% isopropanol. The RNAclear XP beads were eluted with 10μl poly-adenylation mix from the SMARTer smRNA-Seq kit (Takara 635029) after washing. Other steps of the product manual were followed to finish the library preparation with 14 PCR cycles for library amplification.