Illumina NovaSeq 6000 paired end sequencing; Single-cell RNA sequencing of lck-GFP transgenic zebrafish
Single-cell RNA sequencing of lck-GFP transgenic zebrafish
Sample 1_lck_control_p
The generation of germ-free Tg(lck:GFP) zebrafish followed previously established protocols [1]. In brief, 2 hpf embryos were transferred to petri dishes with sterile E3 medium, supplemented with ampicillin (100 μg/mL), kanamycin (5 μg/mL) and amphotericin B (250 ng/mL) and incubated at 28°C. At 50% epiboly up to shield stage (6 hpf), the embryos were surface disinfected with 0.1% of polyvinylpyrroidone-iodine (PVP-I) for exactly 2 min, followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium, transferred to flasks and incubated at 28°C. The viability was monitored, and sterile medium was refreshed daily. At day 4, the hatched embryos were used for the sterility validation. A day 5, germ-free Tg(lck:GFP) zebrafish were exposed to the combination of GO (30 µg/mL) and butyrate (2.5 mM) for 24 h. GO and BA were pre-incubated for 1 h prior to the exposure. Fifty larvae were used as one replicate, four replicates, i.e. two hundred larvae in total, were used for each condition. After the exposure, zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly, zebrafish larvae were euthanized with 0.01% of tricaine for 5 min, collected in 1.5 mL tube, and washed with PBS for three times. The dissociation was initiated by adding 500 μL of pre-warmed enzyme mix containing 460 μL of 0.25% trypsin-EDTA (Gibco) and 40 μL of collagenase (Sigma-Aldrich) at the concentration of 100 mg/mL, followed by mechanical dissociation using P1000 and then P200 pipette tips in heat-block at 30 °C until the tissue was no longer visible (about 10 min). The dissociation was then stopped by adding 800 μL DMEM (Corning Cat# 10-013-CV) with 10% FBS (Fisher Scientific). The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT, followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 μm nylon mesh, with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non-viable cells (Invitrogen Cat# D15106) at the dose of 3 μM for 10 min at room temperature before the fluorescence-activated cell sorting (BD FACSAria III, BD Biosciences, NJ, USA). The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 °