title: s_2cell.APH.WE
organism: Mus musculus
description: Protocols: Growing oocytes were obtained from 12-14-day-old B6D2F1 mice (SLC Japan). The ovaries were transferred to HEPES-buffered KSOM and punctured with a 30-gauge needle. Liberated oocytes with attached follicle cells were harvested and the follicle cells were gently removed using a narrow-bore glass pipette. The oocytes were then transferred into alpha-minimal essential medium (alpha-MEM; Life Technologies, Inc., Grand Island, NY) containing 5% fetal bovine serum and 10 ng/ml epidermal growth factor (both from Sigma-Aldrich). For DRB treatment, 1-cell embryos were transferred into KSOM containing 120 uM DRB (Sigma-Aldrich) or 3 ug/ml aphidicolin (Sigma-Aldrich) at 4 (before initiation of transcription (Aoki et al., 1997)) or 16 h post-insemination ( embryos enter M phase at 16 h after insemination), Total RNA was extracted from 3000, 3000, 4500, 2800, 1400 and 700 MII eggs, 1-, 2-, and 4-cell embryos, morulae and blastocysts obtained from C57BL6/J mice, respectively, using Isogen (Nippon Gene, Tokyo, Japan), according to the manufacturers instructions. In the second trials for MII eggs and 1-cell embryos, similar numbers of cells used for the first trials were used for RNA extraction. RNA quality analysis and size-selection (>200 tn) were performed on the Bioanalyzer RNA Pico Chip (Agilent Technologies, Santa Clara, CA). RNA-Seq libraries were constructed using the mRNA-Seq Sample Preparation Kit (Illumina, San Diego, CA) without selection of polyadenylated RNA. Briefly, size-selected total RNA was fragmented to 40~900 nt fragments in fragmentation buffer at 94 oC for 5 min, reverse transcribed with random primers and ligated with adaptors. cDNA templates were amplified by PCR in the conditions as follows: the initial heat treatment at 98oC for 30 s followed by 15 cycles of 98oC for 10 s, 65oC for 30 s, 72oC for 30 s, and final treatment of 72 oC for 5 min. RNA-seq libraries were subjected to 76-nt paired-end-sequencing.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2015-03-19T17:02:31Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T21:13:23Z
attributes.tag: External Id
attributes.value: SAMEA2781638
attributes.tag: INSDC center name
attributes.value: Faculty of Science, University of Zagreb
attributes.tag: INSDC first public
attributes.value: 2015-03-19T17:02:31Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T21:13:23Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2950:s_2cell.APH.WE
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: cleavage 2-cell
attributes.tag: sample name
attributes.value: E-MTAB-2950:s_2cell.APH.WE
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: strain
attributes.value: C57BL6/J