GSM2574734: RNA_B6/PWK_E65_VE_2; Mus musculus; RNA-Seq
MII oocytes were collected from 8 week-old superovulated BDF1 females and inseminated with BDF1 sperm. At 7 hpf, zygotes were transferred into M2 media containing 5 μg/ml cytochalasin B, and parental pronuclei were exchanged by using a Piezo impact-driven micromanipulator. The sendai virus (HVJ, Cosmo-bio) was used for fusing karyoplasts with cytoplasms as previously described 44. After reconstruction, embryos were cultured in KSOM. When collecting embryos for RNA-seq or/and liDNase-seq, we removed zona pellucida by a brief exposure to Acid tyrode's solution (Sigma-Aldrich), then the embryos were washed first with M2 media, and then 0.2% BSA/PBS. For liDNase-seq, 10 embryos were transferred into an Eppendorf LoBind 1.5 ml tube, and placed on ice until DNase I treatment. For RNA-seq, seven to ten embryos were transferred into a thin-walled RNase-free PCR tubes (Ambion). The 2-cell and morula embryos were collected at 30 and 78 hpf, respectively. When preparing α-amanitin treated 2-cell embryos, zygotes were transferred into KSOM containing 25 μg/ml α-amanitin (Sigma-Aldrich) at 5 hpf and cultured until sample collection at 30 hpf in the presence of α- amanitin. Low-input DNase-seq libraries were prepared as previously described with minor modifications 6. Embryos or nuclei collected in 1.5 ml tubes were resuspended in 36 μl lysis buffer (10 mM Tris- HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Triton X-100) and incubated on ice for 5 min. DNase I (10 U/μl, Roche) was added to the final concentration of 80 U/ml (for the GV nucleus sample) or 40 U/ml (for all the other samples) and incubated at 37 °C exactly 5 min. The reaction was stopped by adding 80 μl Stop Buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.15% SDS, 10 mM EDTA) containing 2 μl Proteinase K (20 mg/ml, Life technologies). Then 20 ng of a circular carrier DNA [a pure plasmid DNA without any mammalian genes purified with 0.5x Beckman SPRIselect beads (Beckman Coulter) to remove small DNA fragments] was added. The mixture was incubated at 50 °C for 1 hr, then DNA was purified by extraction with phenol- chloroform and precipitated by ethanol in the presence of linear acrylamide (Life technologies) overnight at -20 °C. Precipitated DNA was resuspended in 50 μl TE (2.5 mM Tris, pH 7.6, 0.05 mM EDTA), and the entire volume was used for sequencing library construction Sequencing library was prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufactures' instruction with the exception that the adaptor ligation was performed with 0.03 μM adaptor in the ligation reaction for 30 minutes at 20 °C and that PCR amplification was performed using Kapa Hifi hotstart readymix (Kapa Biosystems) for 8-cycles. The PCR products were purified with x1.3 volume of SPRIselect beads (Beckman Coulter) and then size selected with x0.65 volume followed by x0.7 volume of SPRIselect beads and eluted in 24 μl TE. The number of cycles for the second PCR amplification was measured by qPCR using 1 μl of the 1:1,000 diluted eluent. The remaining 23 μl of the samples was then amplified with Kapa Hifi hotstart readymix (we used 7 cycles for all samples in this study). The PCR product was purified with x1.3 volume of SPRIselect beads and then size selected with x0.65 volume followed by x0.7 volume of SPRIselect beads. The DNA was eluted in 30 μl of TE. The libraries were sequenced on a Hiseq2500 with single-end 100 bp reads (Illumina).  RNA-seq libraries were prepared as previously described 17. Briefly, reverse transcription and amplification were performed using whole embryo lysates with SMARTer Ultra Low Input RNA cDNA preparation kit (Clontech). When processing 2-cell AG, PG and α-amanitin-treated IVF embryo samples, 1 μl of 1:40,000 diluted ERCC (External RNA Controls Consortium) standard RNA (Life technologies) was added to each of the tubes at the step of cell lysis. cDNAs were then fragmented using the Covaris M220 sonicator (Covaris). The fragmented cDNAs were end- repaired, adaptor ligated and amplified using NEBNext Ultra DNA Library Prep Kit for Illumina according to the manufacturer’s instruction (New England Biolabs). Single end 100 bp sequencing was performed on a HiSeq2500 sequencer (Illumina).