GSM7157481: Early 4 cell stage embryo Dnmt3a f/- ; Dnmt3b f/- ; Stra8-iCre tg sperm derived rep 1; Mus musculus; RNA-Seq
To obtain early embryos, 7-10-week-old JF1 female mice were injected intraperitoneally with HyperOva (Cosmo Bio, KYD-010-EX-X5) 63 hours pre collection and human chorionic gonadotropin (hCG, Chorulon MSD) 15 hours pre collection. Oviducts were dissected and oocytes from 2 animals were collected in 30 μl drops of HTF medium (EmbryoMax® Human Tubal Fluid Millipore, MR-070-D). Sperm was collected from 2 cauda epididymides in 50 μl drops of HTF medium. 2-5 μl of sperm sample was transferred to the oocyte drops and incubated for 3 hours at 37 °C. Zygotes with visible pronuclei washed 3 times in HTF medium and transferred to KSOM medium (Millipore, MR-106-D) and cultured in low oxygen chamber filled with the mixture gas (5% O2/ 5% CO2). Early 4 cell stage embryos were collected 50-55 hours post fertilization. Each 4-cell stage embryo was collected at 50-55 hours after IVF, was washed in PBS + 0,02% PVA,  transferred into a well of a 96-well plate containing SS2 Lysis buffer ( 0,09% Triton-X 100, 0,5U/μl SUPERAseIN (Life Technologies AM2696), 2.5mM oligodT primer (Microsynth), 2.5mM dNTP mix(Promega), ERCC RNA Spike-In Mix (1:3.2x107 Thermo Fischer scientific 4456740)), snap frozen on dry ice and then stored in -80°C until further use. The sample was incubated at 72°C for 3 minutes and immediately put back on ice. Then SS2 RT mix (10U/µl SuperScript II reverse transcriptase (Thermo Fischer 18064014), 0.25U/μl SUPERAseIN,  1X SuperScript II first-strand buffer , 5mM DT, 1M Betaine (Sigma B0300-1VL), 6mM MgCl2, 1mM TSO (Exiqon)) was added to the sample and incubated for 90 minutes at 42°C, then 10 cycles of 2 minutes at 50°C and 2 minutes at 42°C and finally for 15 minutes at 70°C. Sample was preamplified for 16 cycles using 1X KAPA HiFi HotStart ReadyMix (KAPA Biosystems KK2602) and 0.1µM ISPCR primers (Microsynth). DNA was purified using 1X volumes of Ampure XP DNA purification beads. 1ng of pre-amplified DNA was added to Tn5 tagmentation mix (1x TAPS-DMF buffer, homemade Tn5 (1:1,200)) in total volume 20μl and incubated at 55°C for 7minutes. Then the reaction was stopped by adding 5μl of 0.2% SDS and kept at 25°C for 7 min. Adapter-ligated fragment amplification was done using Nextera XT index kit (Illumina) in a total volume 50μl (1x Phusion HF Buffer, 2 µl of Phusion High Fidelity DNA Polymerase (Thermo Fischer, F530L), dNTP mix (0.3mM each) (Promega)) with 10 cycles of PCR. Library was purified by 1X volumes of Ampure XP DNA purification beads and resuspended in 25 μl nuclease free H2O.