Single-Cell RNA Sequencing

Single-cell transcriptome information was captured (from 20 sample sources) using the BD Rhapsody system. The single-cell suspension was randomly assigned to 200,000 micropores by a limited dilution method. The beads with oligonucleotide barcodes were added to the saturated state and paired with cells in the micropores. The cells were cleaved in micropores to hybridize mRNA molecules and the oligonucleotides on the beads were captured by bar code. Then reverse transcription and ExoI digestion were performed in a test tube. During cDNA synthesis, a unique molecular identifier (UMI) was bound to each cDNA molecule at the 5’ end to indicate the origin of the cell. The BD Rhapsody single-cell full transcriptome workflow includes random primer and extension (RPE), RPE amplification PCR and WTA index PCR to prepare a full transcriptome library. The high-sensitivity DNA chip (Agilent) on the bioanalyzer 2200 and the qubit high-sensitivity DNA analysis (Thermo Fisher Scientific) were used to quantify the library. Sequencing was performed by illumina sequencer (Illumina, San Diego, CA) on a 150 bp paired-end run.