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Cells were passed through a sterile 40 µm cell strainer and centrifuged at 300 g, 4°C for 5 minutes. Supernatant was removed while ensuring the cell pellet was not exposed to the air, and 1 ml RNase-free 3X phosphate buffer solution (PBS) was added to resuspend the cells. We repeated centrifugation and resuspension with 200 µl RNase-free 3x PBS to clean the cells, and a suspension sample was visually inspected under a microscope to assess cell density and viability using trypan blue. Cell density was adjusted to 2×105 cells per ml prior to loading onto a microwell chip (Singleron Biotechnologies). Barcoding Beads (Singleron Biotechnologies) were subsequently collected from the microwell chip, followed by reverse transcription of captured mRNA, and the complementary DNA (cDNA) was further amplified using PCR amplification, fragmented, and then ligated to sequencing adapters according to the GEXSCOPE® Single Cell RNA Library Kit. Individual libraries were diluted to 4 nM, pooled, and sequenced on an Illumina NovaSeq 6000 platform (Illumina) as 167 bp paired end reads. |
RNA-Seq |
TRANSCRIPTOMIC SINGLE CELL |
RANDOM |
PAIRED
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