Illumina HiSeq 4000 paired end sequencing; Tracing the emergence of primordial germ cells from bilaminar disc rabbit embryos and pluripotent stem cells (embryo single cell RNA-sequencing)
Tracing the emergence of primordial germ cells from bilaminar disc rabbit embryos and pluripotent stem cells (embryo single cell RNA-sequencing)
OC049_p
Rabbit embryos at E6-7 were trypsinized with 0.25% trypsin/EDTA for 5min. Then, single cells were sorted by SH800 into wells of 384 well plates filled with 2.875 ul of lysis buffer (Takara Bio Inc.). We had also included additional samples; cells from posterior epiblast at E7 containing rbPGCs, in vitro rbPSCs, rbPSCs cultured for 2 days with lower concentration (5ng/ml) of FGF (termed as rbPSC_F5), and NT positive d2 rbPGCLCs. To enrich rbPGCs, 3-4 posterior epiblast dissected from E7 (st.3-4) rabbit embryos were dissociated into single cell by TrypLE Express (Thermo Fisher Scientific) at 37℃ for 8 min. After inactivation of the TrypLE Express, dissociated cells were incubated with anti-c-KIT antibody labeled by Biotin Labeling Kit (Dojindo), and subsequently incubated with Alexa Fluor® 647 Streptavidin (Biolegend, San Diego, CA). For rbPSC rbPSC_F5 and rbPGCLC samples, cells dissociated with 0.25% trypsin/EDTA (Thermo Fisher Scientific) were incubated with anti-feeder cell antibody (Miltenyi Biotec) for 15 min at 4℃ to exclude residual MEF by FACS. cells were directly sorted into PCR tubes filled with 10 ul of the lysis buffer by SH800 or MA900 cell sorter. Single-cell transcriptomes of sorted cells were assayed as follows: for E6-7 embryos, reverse transcription and cDNA amplification was performed using the miniaturized version of the SMART-seq HT protocol using the low-volume liquid-handling robot Mosquito HV (SPT Lab Tech). For additional samples, reverse transcription and cDNA amplification was performed according to manufacturer's protocol of Smart-seq HT kit (Takara Bio Inc.).  Libraries were prepared using the Illumina Nextera XT DNA preparation kit.