GSM3728973: t05_d5cpc; Homo sapiens; RNA-Seq
Specific day time point of selected six different stages are day 0 (hESC), day 2 (MES), day 5 (CP), day 9 (CM), day 14 (immature CM), day 60(mature CM). Above stage cells were digested into single cell suspension. After digestion, and then filtered through a 70μm cell strainer into 15ml tubes. Single-cell sequencing library was generated by use of iCell8 platform (Takara). In general, isolated cells were stained with a mixture of Hoechst 33342 and Propidium Iodide (R37610, Thermo Scientific) according to the manufacturer's instruction. After staining, cells were washed by PBS, and counted by MoxiTM Automated Cell Counter. Afterwards, cell suspension (20,000 cells/ml) was submitted to the MultiSample NanoDispenser (MSND, Wafergen Biosystems) for single cell preparation. The dispensed cells were then imaged with the Imaging Station, and single live cells, defined by Hoechst- positive and Propidium Iodide-negative staining, were selected. Selected cells were subjected to reverse transcription and first-step amplification in a Chip Cycler (Bio-Rad), and the resulting cDNA was purified and size-selected with Agencourt AMPure XP beads (A63880, Beckman Coulter) One ng of purified cDNA was applied to generate a sequencing library by use of Nextera XT DNA sample preparation Kit (FC-131-1024, Illumina). Libraries were sequenced on the NextSeq500 sequencer (Illumina) using the 26nt and 50nt paired-end sequencing protocol.