4 protocols
AccessionType
nucleic acid sequencing protocol
mRNA from the single blastomeres was amplified using the SMARTSeq2 protocol, with the additional inclusion of ERCC spike-in control (1 l of 1:250,000 dilution of mix 1 (Ambion) per cell). Multiplex sequencing libraries were generated from amplified cDNA using Nextera XT (Illumina) and sequenced on a HiSeq 2500 running in rapid mode.
nucleic acid library construction protocol
Multiplex sequencing libraries were generated from amplified cDNA using Nextera XT (Illumina) and sequenced on a HiSeq 2500 running in rapid mode.
sample collection protocol
Embryos were recovered from superovulated F1 (C57Bl6xCBA) mice into M2 media supplemented with 4% BSA, as described previously25. Individual blastomeres were collected at late 2-cell (48 hours after hCG), late 4-cell (10 hours after the first 2-cell blastomere divided), late 8-cell (10 hours after the first 4-cell blastomere divided), 16-cell (78 hours after hCG) and 32-cell (86 hours after hCG) stages. Embryos for blastomere isolation at the 2-, 4,- and 8-cell stages were all collected at the 2-cell stage and cultured in KSOM media (Millipore) at 37C with 5% CO2. Embryos for the 16- and 32-cell blastomeres were collected directly at the respective stages. The zona pellucida of individual embryos was removed using Tyrode's solution (Sigma). Zona-free embryos were incubated for 5 minutes (for the 2,4 and 8-cell stages) or 20 minutes (for the 16 and 32-cell stages) in Ca2+ and Mg2+ free M2 before disaggregation by careful pipetting with a flame-polished glass pipette. Each embryo was processed individually. Single blastomeres for all stages were placed into individual tubes containing 2.3 l of 0.2 % Triton X-100 (Sigma) supplemented with 1 U/l RNAsIN (Ambion).
sample collection protocol
At the late 2-cell stage one blastomere was injected with rhodamine-dextran (Invitrogen), as described previously. Embryos were observed at 20 minute intervals to categorise the order of divisions and whether each cleavage was meridional (M) or equatorial (E), relative to the position of the second polar body. 4-cell stage embryos were sorted into ME, EM, EE and MM groups. Embryos in which the polar body was absent were discarded. Following disaggregation, the individual cells were inspected under an epifluorescent inverted microscope and classified as originating from the first- or second-dividing 2-cell stage blastomere based on the rhodamine fluorescence.