GSM7985977: scRNA-seq_hmSpVO_D30; Homo sapiens; RNA-Seq
For single cell RNA sequencing, hdMOs from early stage group (day 30) and late stage group (day 62) were collected and dissociated into single cells. Briefly, media was removed on a centrifuge machine at 500g for 5 min and then 1 ml dispase was added, the organoids were lightly cut with a scissor, digested in metal bath at 37℃ for 30min. The suspension was centrifuged at 500g for 5min to remove supernatant and 1ml trypsin was added to continue digesting at 37 degrees for 15-30min. After completely digested, cells were passed through a 40um sieve and washed with DPBS+10% BSA 2-3 times for further experiments.For the quality control and counting of single cell suspension, the cell survival rate is generally above 80%. Cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) at a concentration of 1,000 cells per μl (Single Cell 3′ library V3) as described in the manufacturer's protocol. GEMs (Gel Bead in Emulsion) were constructed for single cell separation according to the number of cells to be harvested. After GEMs were normally formed, GEMs were collected for reverse transcription in a PCR machine for labeling. Then the GEMs were oil-treated, and the amplified cDNA was purified by magnetic beads, and then subjected to cDNA amplification and quality inspection. The 3ʹ Gene Expression Library was constructed with the quality-qualified cDNA. After fragmentation, adaptor ligation, sample index PCR, etc., the library is finally quantitatively examined and was sequenced on the Illumina Novaseq 6000 instrument using 150-base-pair paired-end reads.