RNA sequencing of zebrafish asxl1+/+ and asxl1-/-

ASXL1 gene is one of the most frequently mutated genes in malignant myeloid diseases. In patients, ASXL1 mutations are usually heterozygous frameshift or non-sense mutations leading to C-terminal truncation. Here, we generated an endogenous C-terminal truncated Asxl1 mutant in zebrafish which is more comparable to human malignant leukemia patients. Our data showed that at embryonic stage, neutrophil differentiation was explicitly blocked in our mutant. To understand the basis for the impairment of neutrophil differentiation in zebrafish asxl1 mutants, we performed RNA-seq of asxl1 mutants at 3dpf and their littermate controls. Similar with the phenotype we observed, the expression of neutrophil markers were all included in down-regulated genes. Nonetheless, the expression of myeloid progenitor marker and macrophage marker were not impaired in asxl1 mutants. We also found inflammatory cytokine and matrix metalloproteinases were upregulated after mutated asxl1. It suggests that neutrophil deficiency may stimulate the expression of some inflammatory cytokines and enhances the inflammatory responds. Therefore, transcriptome analysis mainly represented the disruption of neutrophil development. 3 days post fertilization (dpf) asxl1+/+ and asxl1-/- embryos from asxl1+/- intercross were collected and extract total RNA from the tails including CHT. Each sample is a pool of tails (about 20). Sequencing libraries were prepared from total RNA using Novogene NGS RNA Library Prep Kit. Sequencing was done on an Illumina NovaSeq sequencing platform to obtain 150bp 2 ´ 150 bp