GSM8403315: Liver_Control2; Danio rerio; RNA-Seq
Zebrafish were euthanized in 500 mg/l solution of tricaine (MS-222) (Sigma-Aldrich, E10521). The entire liver was dissected out of the body using Dumont #5 forceps (Fine Science Tools, 11295-10). All steps for making the single-cell suspension were performed with tubes and pipette tips coated with 1% BSA in PBS prior to the sample preparation. Briefly, the liver was dissociated into single cells by incubation it in TrypLE (Thermo Fisher, 12563029) at 37°C in a benchtop shaker set at 1000 rpm for 15 min. Following dissociation, TrypLE was inactivated with goat serum. To remove undissociated chunks and debris, the solution was passed through a 40 μm cell strainer (Miltenyi Biotec, 130-041-407). Cells were pelleted by centrifugation at 500 g for 5 min at 4°C with soft-stop setting. The supernatant was carefully discarded and the pellet re-suspended in 500 μl of PBS. To remove dead cells, Calcein violet (Thermo Fisher, C34858) or Draq7 (Thermo Fisher, D15105) was added at a final concentration of 1 µM and 3 µM, respectively, and the cell suspension was incubated at room temperature for 20 min. The single-cell preparation was sorted using appropriate gates, including excitation with UV (405 nm) or a 633 nm laser for identifying live cells with Calcein+ or Draq7-, respectively. FACS was performed  using a 100 µm nozzle, and a minimum of ~15,000 live cells per condition were collected into BSA-coated tubes containing 1% BSA in PBS. Sorting time did not exceed 15 min. For single-cell RNA sequencing using the 10x Genomics platform, the cell suspension was adjusted with PBS to a density of 300 cells/µl and diluted with nuclease-free water according to the manufacturer's instructions to yield 7,000 cells. Subsequently, the cells were carefully mixed with reverse transcription mix before loading the cells on the 10x Genomics Chromium system (Single Cell 3' v3). After the gel emulsion bead suspension underwent the reverse transcription reaction, emulsion was broken and DNA purified using Silane beads. The complementary DNA was amplified with 12 cycles, following the guidelines of the 10x Genomics user manual. The 10x Genomics single-cell RNA-seq library preparation—involving fragmentation, dA tailing, adapter ligation, and indexing PCR—was performed based on the manufacturer's protocol. After quantification, the libraries were sequenced on an Illumina NextSeq 550 machine.