Project description:Zebrafish eggs were collected at 28 °C, and split into three embryonic incubation temperature groups (24 °C, 28 °C, 32 °C). When larvae reaching first feeding, the incubation temperature of larvae from 24 °C and 32 °C was gradually changed to 28 °C, and all fish were kept at 28 °C for later development until adulthood. At 100 d post-fertilization, 7 replicates from each temperature group were intraperitoneally (i.p.) injected with 2 μl of 50 mg/ml lipopolysaccharide (LPS) from Pseudomonas aeruginosa 10, while another 7 replicates were i.p. injected with 2 μl phosphate-buffered saline as control. At 12 h post-injection, the spleen was dissected, and total RNA was extracted. Five LPS-treated replicates and five controls were used for building RNA libraries and sequencing. The differential gene expression analysis were performed between fish from different embryonic incubation temperatures (24 °C vs 28 °C; 32 °C vs 28 °C), and between LPS treatment and control within each temperature group (24 °C LPS vs control; 28 °C LPS vs control; 32 °C LPS vs control). Totally, 251 differentially expressed genes (DEGs, 71 up-/180 down-regulated) were identified in fish from 24 °C embryonic incubation temperature compared to fish kept at constant 28 °C (DESeq2, adjusted p-value < 0.05, |fold change| > 1.5); and 660 DEGs (385 up-/275 down-regulated) were identified in fish from 32 °C embryonic incubation temperature compared to fish kept at 28 °C. By comparing LPS-treated fish to control, 567 DEGs (271 up-/296 down-regulated) were identified in fish from embryonic incubation temperature of 24 °C, 140 DEGs (80 up-/60 down-regulated) were identified in fish kept at constant 28 °C; and 49 DEGs (11 up-/38 down-regulated) were identified in fish from the 32 °C embryonic incubation temperature group.
Project description:Zebrafish eggs were collected at 28 °C, and split into three temperature groups (24 °C, 28 °C, 32 °C) for incubation. When larvae reaching first feeding, the incubation temperature of larvae from 24 °C and 32 °C was gadually changed to 28 °C, and all fish were kept at 28 °C for later development until adulthood. At 100 d post-fertilization, seven replicates from each group were intraperitoneally (i.p.) injected with 2 μl of 50 mg/ml lipopolysaccharide (LPS) from Pseudomonas aeruginosa 10, while another seven replicates were i.p. injected with 2 μl phosphate-buffered saline as control. At 12 h post-injection, the spleen was dissected, and total RNA was isolated. Five LPS-treated replicates and five controls were used for building small RNA libraries and sequencing. A total of 112 novel miRNAs were identified using miRDeep2. By comparing fish from different embryonic incubation tmeperatures, 32 miRNAs (29 up-/3 down-regulated) were identified differentially expressed (DEmiRs, adjusted p-value < 0.05, |fold change| > 2.0, DESeq2) in fish from embryonic incubation temperature of 32 °C compared to fish kept at constant 28 °C, 3 DEmiRs (2 up-/1 down-regulated) were identified in response to LPS in fish kept at 28 °C. A total of 9,116 target genes were predicted using miRanda (v3.3a), including 8,224 genes targeted by 32 DE miRNAs and 892 genes targeted by 3 DE miRNAs.
