GSM7027996: D3_siNC_rep1; Mus musculus; RNA-Seq
Injected embryos were cultured in KSOM medium under mineral oil in 35 mm petri dishes (Corning Life Sciences, 430165) in the humidified multi-gas incubator (5% O2, 6% CO2 and 89% N2) at 37°C.Embryos were obtained and placed into a 0.2-ml thin-walled PCR tube containing 2 µl of cell lysis buffer (with 5% RNase inhibitor and 95% Triton X-100), 1 µl of 10 µM oligo-dT primer and 1 µl of dNTP mix (10 mM each; Fermentas, R0192). The mix was quickly vortexed and then span down at 700g for 10 s at room temperature) and immediately transferred on ice. The samples were incubated at 72°C for 3 min and immediately put back on ice. 5.7 µl of the RT mix containing SuperScript II reverse transcriptase (Invitrogen, cat. no. 18064-014), RNAse inhibitor (10U), 5X Superscript II first-strand buffer, 100mM DTT (Invitrogen, 18064-014), 5M Betaine (BioUltra ≥99.0%; Sigma-Aldrich, 61962), 1M MgCl2, 100 µM TSO were mixed by gently pipetting up and down. The sample was incubated at 42°C for reverse transcription, and then amplified with First-strand reaction KAPA HiFi HotStart ReadyMix (KAPA Biosystems, KK2601). The cDNA was used for library construction using Vazyme TruePrep DNA Library Prep Kit V2 for Illumina kit (TD503-01)