RNA-Seq of homo sapiens: adult knee synovial tissues
After acquiring the synovial cell suspensions, beads with unique molecular identifier (UMI) and cell barcodes were loaded close to saturation to ensure that each synovial cell was paired with a bead in a Gel Beads-in-emulsion (GEM). After cell lysis, polyadenylated RNA molecules were hybridized to the beads. Then the beads were retrieved into a single tube for reverse transcription. During the synthesis of cDNA, each cDNA molecule was tagged with UMI and cell labels on the 5end (that is, the 3end of a messenger RNA transcript), indicating its cell origin. Briefly, 10 beads then went through procedures including second-strand cDNA synthesis, adaptor ligation, and universal amplification. Furthermore, sequencing libraries were prepared by using randomly interrupted whole-transcriptome amplification products to enrich the 3 end of the transcripts linked with the cell barcode and UMI. The remaining procedures, including the library construction, were performed according to the standard manufacturers protocol (CG000206 Rev D). Sequencing libraries were quantified by utilizing the High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 and the Qubit High Sensitivity DNA Assay (Thermo Fisher Scientific). The libraries were finally sequenced on NovaSeq6000 (Illumina) with 2x150 chemistry.