GSM8172114: Si8, replicate 1, scRNAseq; Homo sapiens; RNA-Seq
Pre-experiment utilizing Trypan Blue staining was conducted to ensure the viability of wtMCF7 cells was above 90% and cell was in optimal growth condition with confluency requirement of over 80%. Subsequently, the culture medium was washed off using Dulbecco's PBS (DPBS) buffer solution without calcium and magnesium, followed by digestion with 0.25% EDTA trypsin at 37℃ for 3 minutes. The detached cells were then collected by centrifugation at 500g for 5 minutes after being transferred to a centrifuge tube using culture medium. After resuspending the cells in DPBS buffer solution and performing cell counting, cells were finally diluted to density of 10e6 per milliliter. Once the cell preparation was completed, it needed to be immediately placed on ice for subsequent library construction using the 10xgenomics platform. Frozen-stored cells were resuscitated rapidly and examined by microscope after 0.4% Trypan blue coloring. When the viability of cells was higher than 80 percent, the experiment of library construction was performed. Briefly, single cells, reagents and Gel Beads containing barcoded oligonucleotides were encapsulated into nanoliter-sized GEMs (Gel Bead in emulsion) using the GemCode Technology. Lysis and barcoded reverse transcription of polyadenylated mRNA from single cells were performed inside each GEM. Post RT-GEMs were cleaned up and cDNA were amplified. cDNA was fragmented and fragments end were repaired, as well A-tailing was added to the 3' end. The adaptors were ligated to fragments which were double sided SPRI selected. Another double sided SPRI selecting was carried out after sample index PCR. The final library was quality and quantitated in two methods: check the distribution of the fragments size using the Agilent 2100 bioanalyzer, and quantify the library using real-time quantitative PCR (QPCR) (TaqMan Probe). The final products were sequenced using the DNBSEQTMplatform.