Transcriptomic Profiling of Zebrafish Intestine and Liver

The intestine tissue or liver bud were dissected from 5 dpf-old zebrafish embryos. Intestine tissues from ~30 embryos or liver buds from ~50 embryos were pooled and then treated with TRIZOL for total RNA extraction. The micro-dissected intestine was also cut into three fragments corresponding to the anterior, middle and posterior portions of the intestine, and the fragments were pooled for total RNA extraction. The obtained total RNA (~200–400 ng/sample) was used for library construction via the Smart-seq2 protocol as described in [24 (link)]. Library sequencing was performed on an Illumina HiSeq X Ten PE150 platform (Illumina, San Diego, CA, USA). RNA sequencing reads were aligned with HISAT2 (version 2.1.0) to the zebrafish genome (GRCz11) and ENSEMBL v99 annotations [25 (link),26 (link),27 (link)].
Analysis of the differentially expressed genes (DEGs) was conducted with DESeq2 (v1.28.1) and gene ontology (GO) set overrepresentation analysis was performed with ClusterProfiler (v3.16.1) [28 (link),29 (link)]. TFBS analysis for the identification of TF regulators was performed by David Bioinformatics Resources [30 (link)].