GSM5827306: CTumor4; Homo sapiens; RNA-Seq
single-cell suspensions of the collected samples were prepared through mechanical dissociation and enzymatic degradation within 2-3h after surgery. Each cell suspension was performed to 3' single-cell RNA sequencing using Single Cell A Chip Kit, Single Cell 3' Library and Gel Bead Kit V2, and i7 Multiplex Kit (10× genomics, Pleasanton, CA, USA) with a cell recovery target of 10000, following the manufacturer's instructions. After generation of GEMs, reverse transcription reactions used barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with Dyna-Beads Myone Silane Beads (Beads, 10× genomics, Cat# 2000048). Then, the cDNA was amplified by PCR with appropriate cycles, which depended on the recovery of cells. Subsequently, the amplified cDNA was fragmented, end-repaired, A-tailed, Index adapter ligated, and library amplification. Qualitative and quantitative assessments of the final libraries were performed on a 12-channel Fragment Analyzer with the High Sensitivity NGS Fragment 1-6000 bp assay (Agilent, Santa Clara, CA), and the Qubit 3.0 Fluorometer with the 1× dsDNA High Sensitivity kit (Life Technologies, Carlsbad, CA). Finally, these libraries with an average final concentration of ~30nM and fragment size of ~450bp were then normalized, pooled and sequenced on the NextSeq 500 using a 150 cycle NextSeq 500 high Output Kit v2.5 (Illumina, San Diego, CA). In addition, 150bp paired-end reads were generated.