GSM8291615: ozt_S1; Homo sapiens; RNA-Seq
Single-cell RNA sequencing (scRNAseq) was conducted on primary human liver cell populations obtained from the healthy donor UBV. The mixture of hepatic cell populations were carefully washed in Dulbecco's phosphate-buffered saline (DPBS, 1X; Corning, Cat No. 21-031-CV) and resuspended at a concentration of 10^6 cells/mL to prevent cell aggregates. The scRNAseq protocol utilized the 10x Genomics Chromium controller and the Chromium single-cell 3' library and gel bead kit (10x Genomics, Cat No. PN-1000075) following the standard manufacturer's protocols. Briefly, gel beads-in-emulsion (GEMs) were generated by combining barcoded single-cell 3' v3 gel beads, a master mix containing cells, and partitioning oil onto Chromium chip B. Cells were delivered at a limiting dilution to achieve single-cell resolution, with the majority (~ 90–99%) of generated GEMs containing no cells. Between 2,000–21,000 live cells were loaded onto the Chromium controller to recover between 1,500 − 15,000 cells for library preparation and sequencing. Following GEM generation, the gel beads were dissolved, primers were released, and any co-partitioned cells were lysed.   An Illumina TruSeq Read 1, 16 nt 10x barcode, 12 nt unique molecular identifier (UMI), and 30 nt poly(dT) sequence were mixed with the cell lysate and a master mix containing reverse transcription (RT) reagents. Incubation of the GEMs produced barcoded, full-length cDNAs from poly-adenylated mRNAs. Subsequently, GEMs were broken, and cDNA was amplified and quantified using an Agilent high sensitivity DNA screentape. SPRIselect magnetic beads were used to purify the first-strand cDNA, followed by PCR amplification to generate sufficient mass for library construction. Enzymatic fragmentation and size selection optimized the cDNA amplicon size, with TruSeq Read 1 added during GEM incubation. P5, P7, a sample index, and TruSeq Read 2 were added via end repair, A-tailing, adaptor ligation, and PCR. Library quality was assessed using an Agilent Bioanalyzer high sensitivity chip. Sequencing was performed on the Illumina HiSeq 2500 with a target of 150,000 reads/cell.