title: GSM4118746: C57/BL6J oocyte cells pacbio; Mus musculus; RNA-Seq
library_construction_protocol: Sperm were collected from the epididymis of DBA/2N males. MII oocytes were derived from 3 to 5 week-old C57BL/6J females induced to superovulate by injection of 7.5IU pregnant mare serum gonadotropin (PMSG, San Sheng), followed by injection of 7.5IU human chorionic gonadotropin (hCG, San Sheng) 48h later. Embryos were collected from the oviduct of the superovulated females after mating them with DBA/2N males. Oocytes and embryos were isolated in M2 medium (Sigma) at 20h post-hCG, then while oocytes were lyse directly, the embryos were not lyse until they develop to defined stages: zygotes (24-26h post hCG), two-cell stage (46-48h post-hCG), four-cell stage (54-58h post-hCG), eight-cell stage (68-70h post-hCG), blastocyst stage (94-96h post-hCG), with culturing in KSOM media (Merck Millipore) at 37℃ with 5% CO2. Embryos were lysed in 50 μl of guanidine isothiocyanate solution (Invitrogen, 15577-018) at 42°C for 15 min. Nuclease-free water was added to 200 μl, and 3 volumes of absolute ethanol, 1/10 volume of 3 M acetate sodium (Invitrogen, AM9740) and 2 μl of glycogen (Roche, 10901393001) were added and mixed uniformly. After treatment at -80 °C for 2 h, total RNA was pelleted by centrifugation at 12,000g for 40 min. Total RNA pellets were dissolved in lysis solution and used as template for the first cDNA synthesis, cDNA was amplified by Smart-seq2 protocol1. 2 μg of each amplified cDNA is was transformed into SMRTbell templates using the PacBio SMRTbell Express Template Prep Kit. The libraries were sequenced using the PacBio Sequel System in Berry Genomics Company (Beijing, China).The sequencing libraries were constructed using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme Biotech, TD502-01) according to the manufacturer's instruction. All libraries were sequenced on Illumina NovaSeq 6000 platform with a 150-bp paired-end module in Berry Genomics Company (Beijing, China).
attributes.tag: GEO Accession
attributes.value: GSM4118746