GSM3288184: H37; Macaca mulatta; RNA-Seq
To collect single blastomeres, embryos with good quality were transferred into 0.1% Pronase(Merck) in PBS to remove zona pellucida, followed by three washes in 0.05% PVP-PBS. To distinguish the outer and inner cells, embryos were incubated with PKH26 dye (Sigma) which labels the live outer cells with red fluorescence. After labeling, the embryos were washed for three times in PVP-PBS and cut off the blastocele with 1 mL syringe. Then the remaining embryo tissues were rinsed with Ca2+/Mg2+ free PVP-PBS and treated with 0.125% trypsin-EDTA for at least 5 min at 37℃ until the cell surface looks rough. Disaggregating was done by a mouth pipette aided by several finely pulled glass capillaries of different internal diameter. After most cells were separated apart, single cells were individually transferred into small droplets of 0.1% BSA-PBS and the inner/outer cell identity was identified by the fluorescent microscopy. Cells showing no fluorescence were inner cells, whereas those with red fluorescence were outer cells. Briefly, single cells were washed three times in BSA-PBS and those with a clear round shape were added into cell lysis buffer, followed by First-strand cDNA synthesis and PCR amplification. PCR products were purified by Ampure XP beads and used for libraries construction. Libraries were deep sequenced on Illumina X-ten platform, using 150-bp paired-end strategy.