GSM2838730: G.HB.I.11; Macaca mulatta; RNA-Seq
To collect single blastomeres, embryos with good quality were transferred into 0.5% Pronase in TL-HEPES to remove zona pellucida, followed by three washes in PVP-BSA. To distinguish the outer and inner cells, embryos were incubated with PKH2 dye (Sigma) which labels the live outer cells with red fluorescence. After labeling, embryos 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 PVP-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 PVP-PBS and those with a clear round shape were added into cell lysis buffer, followed by cDNA synthesis, primer removal, poly(A) tailing, second-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 HiSeq 2500 platform, using 100-bp single-end strategy.
GEO Accession
GSM2838730