GSM9659777: hPCO_D6; Homo sapiens; RNA-Seq
For single-cell RNA sequencing analysis, hPCOs at day 6, day 9, and day 16 were harvested and dissociated into single cells. In brief, 10-12 hCPOs were chopped into 1-mm cubic fragments, followed by enzymatic treatment using a solution of 2 mg/ml collagenase II, 2 mg/ml collagenase IV, and 0.1 mg/ml DNase I, and then incubated on an orbital shaker at 37°C for 30 minutes. The cell concentration was assessed using AO/PI fluorescent staining reagents, with the requirement that the cell viability rate must exceed 80%. The isolated cells were then directly loaded into the 10X Chromium Single Cell Platform (10X Genomics) at a concentration of 1,000 cells per μl, following the guidelines provided in the manufacturer's instructions. GEMs (Gel Bead in Emulsion) were formed for the isolation of single cells based on the anticipated number of cells to be obtained. Once the GEMs were generated, they were gathered for reverse transcription in a PCR thermocycler for labeling purposes. Subsequently, the GEMs were subjected to oil treatment, and the amplified cDNA was purified using magnetic beads, followed by cDNA amplification and quality assessment. The 3′ Gene Expression Library was constructed using the cDNA that had passed the quality control checks. After procedures such as fragmentation, adapter ligation, and sample index PCR, the library was quantitatively measured and sequenced on the Illumina Novaseq 6000 platform with 150-base-pair paired-end reads.