Transcriptome sequencing of hepatocytes (with or without GFP) during liver development in zebrafish
The hepatocytes and non-hepatocytes within the livers of 3dpf, 7 dpf, and adult transgenic Tg(fabp10:GFP) zebrafish were utilized for experimentation. Upon cessation of opercular movements, animals were euthanized via immersion in ice-cold water for ten minutes. In this transgenic line, hepatocytes in the liver express GFP. Subsequently, liver tissues were subjected to enzymatic digestion using 1 mg Collagenase IV dissolved in 1 ml L-15 medium, incubated at room temperature for 20 minutes. Liver tissues were then transferred to Leibovitz L-15 medium with an osmolarity of 300 mOsm and pH adjusted to 7.35. Hepatocytes and non- hepatocytes were isolated through gentle trituration. The chamber was mounted onto the stage of an Olympus inverted microscope equipped with fluorescence capabilities, perfused with fresh L-15 medium for 5 minutes to rinse off debris. Calibrate flow cytometer (Moflo XDP, Beckman), ensuring that the instrument settings (such as voltage thresholds and compensation matrices) are properly configured for GFP detection. Use beads calibrated for green fluorescent protein to adjust the photomultiplier tube voltages and other parameters. Prepare sterile sorting tubes filled with a suitable buffer to collect sorted cells. Pre-chill if necessary. Use an argon laser typically at 488 nm for GFP excitation. Set appropriate filters to detect GFP emission, usually centred around 530 nm. Discriminate intact cells from cell fragments based on size and internal complexity. Balance purity and yield by adjusting sort speed and stringency of gating parameters. Follow strict cleaning protocols after sorting to avoid contamination. Isolated cells were collected utilizing flow cytometry, enabling precise sorting of GFP-positive hepatocytes from GFP-negative non-hepatocytes. Approximately 400 hepatocytes and non-hepatocytes were harvested from around 12 zebrafish, with 3 replicates per sample. The careful isolation procedures and subsequent sorting by flow cytometry facilitated acquisition of pure populations of hepatocytes and non-hepatocytes, ensuring gene expression profiles and reliable downstream analyses of cellular composition.