GSM7157027: Early 2 cell stage embryos H3K4me3 Dnmt3a f/+ ; Dnmt3b f/+ sperm derived  rep 3; Mus musculus; OTHER
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 2 cell stage embryos were collected 22 hours post fertilization. We fused N terminally the Tn5 transposase to two tandem IgG binding Z domains to specifically target the ZZ-Tn5 to antibody bound regions. Early 2 cell embryos (50 embryos per reaction) were incubated in 30 ul LIB buffer (20mM HEPES pH 7.5, 300mM NaCl, 0.5mM Spermidine, 0.05% Digitonin, 0.05% Triton X-100, 1X EDTA-free protease inhibitor cocktail) on ice for 10 mins for nuclei permeabilization. Active transposomes were generated by incubating ZZ-Tn5 (8μM) with Mosaic End double-stranded (MEDS) oligonucleotides (12μM) for 30 minutes at 37°C. Active transposomes (0.4 μM) were incubated with anti-H3K4me3 antibody or normal rabbit IgG (1μM) in LIB buffer for 30 minutes at 4°C with continuous mixing. Antibody- ZZ-transposomes complexes (final concentration 0.1 μM) were added to the embryo samples and incubated for 1 hour at 4°C with continuous mixing to allow targeting to the chromatin. Antibody- ZZ-transposomes were activated by addition of TAB buffer (20mM HEPES, 5%PEG6000, 7.5mM MgCl2, 300mM NaCl) and incubated for 1 hour at 37°C with continuous mixing. The tagmentation reaction was stopped with addition of Stop buffer (0.2% SDS, 10mM EDTA, 100ug/ml ProteinaseK) and incubated at 65°C for 15 minutes. DNA was purified by phenol-chloroform, ethanol precipitation and libraries were amplified for 16 cycles. Libraries were purified by 0.5X-1.5X volumes left-right side of Ampure XP DNA purification beads and sequenced in 50-bp single-end mode with HiSeq 2500 sequencing technology (Illumina), per the manufacturer's recommendations.