title: GSM8186674: D8, scRNA-seq; Homo sapiens; RNA-Seq
library_name: GSM8186674
library_construction_protocol: In hematopoietic induction in vitro, samples of D0, D2, D4, D6, D8, D10 are dissociated into single-cell solution. Briefly, cells from different time point samples were dissociated with 1 ml/well of Accutase at 37°C for 8 min. Cells were washed and resuspended with differentiation medium (IMDM, 5 ng/ml BMP4, 10 ng/ml VEGF, 2 U/ml EPO (MCE), 20 ng/ml SCF (MCE), 15 μM NAC (Sigma-Aldrich), and 1 μM minocycline hydrochloride (Selleck)). The collection of cells was performed using the Dead Cell Removal Kit (Miltenyi). For labeling experiments, transfer the prepared single cells to the labeling culture medium and keep the cell culture away from light. After incubation, discard supernatant and resuspend cell pellet in an appropriate volume of PBS. Glycosylation sites on the cell surface were labeled with fucose residues carrying a tag containing a PCR handle, known sequence, and polyA sequence, and glycosylation sites on the cell surface were labeled with fucose residues carrying a tag containing a PCR handle, known sequence, and polyA sequence. Single-cell suspensions with PBS (HyClone) were loaded onto microwell chip using the Singleron Matrix® Single Cell Processing System. Barcoding Beads are subsequently collected from the microwell chip, followed by reverse transcription of the mRNA captured by the Barcoding Beads and to obtain cDNA, and PCR amplification. The amplified cDNA is then fragmented and ligated with sequencing adapters. The scRNA-seq libraries were constructed according to the protocol of the GEXSCOPE® Single Cell RNA Library Kits (Singleron). Individual libraries were diluted to 4 nM, pooled, and sequenced on Illumina novaseq 6000 with 150 bp paired end reads.