DOI: 10.5061/dryad.jsxksn0gc
title: Bulk RNA-seq of macrophages isolated from Salmonella Typhimurium infected zebrafish embryos

description: Bacterial infection involves a complex interaction between the pathogen
 and host where the outcome of infection is not solely determined by
 pathogen eradication. To identify small molecules that promote host
 survival by altering the host-pathogen dynamic, we conducted
 an in vivo chemical screen using zebrafish embryos and found that
 treatment with 3-hydroxy-kynurenine protects from lethal gram-negative
 bacterial infection. 3-hydroxy-kynurenine, a metabolite produced through
 host tryptophan metabolism, has no direct antibacterial activity but
 enhances host survival by restricting bacterial expansion in macrophages
 by targeting kainate-sensitive glutamate receptors. These findings reveal
 new mechanisms by which tryptophan metabolism and kainate-sensitive
 glutamate receptors function and interact to modulate immunity, with
 significant implications for the coordination between the immune and
 nervous systems in pathological conditions.
description: The transcriptomes of macrophages isolated from zebrafish embryos
 (3 dpf) were analyzed by RNA sequencing. Briefly, fluorescently labeled
 cells were isolated in bulk from a single cell suspension obtained by
 enzymatic digestion of groups consisting of 5 homozygous Tg(mpeg1:mCherry)
 transgenic animals (8-11 biological replicates/condition) following the
 protocol as described in the flow cytometry section. cDNA libraries were
 constructed from a range of 50-800 collected cells with an in-house
 adaptation of the Smart-seq2 protocol in combination with the Nextera XT
 Library preparation kit (Illumina, Inc., San Diego, CA). Library size and
 concentration were evaluated using the TapeStation 2200 system (Agilent)
 and a Qubit fluorometer (Invitrogen) before sequencing. Samples were
 multiplexed and paired-end sequences of 50 bp were generated on a NovaSeq
 S4 6000 sequencing system (Illumina) generating on average 1.03e8 reads
 per sample. Samples were demultiplexed and FASTQ files representing each
 sample were generated. Any remaining adapter sequences were removed using
 skewer (0.2.2) and FASTQ files were assessed for quality control using
 FASTQC (0.11.5). Reads were aligned to the Ensembl zebrafish reference
 genome GRCz11 using Hisat2 (2.1.0) and counts were quantified using
 HTSeq-Count (0.12.4). Differential gene expression analysis was performed
 using DESeq2 (1.28.1) with a significance cut-off of p &lt; 0.05. Gene
 ontology analyses were performed using gene set enrichment analysis (GSEA)
 and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses
 with an FDR below 0.05 using gseGO, gseKEGG, and GSEA, respectively, from
 ClusterProfiler.
description: # Bulk RNA-seq of macrophages isolated from Salmonella Typhimurium
 infected zebrafish embryos From the article: A tryptophan metabolite
 modulates the host response to bacterial infection via kainate receptors
 --- ## Data Descriptions The following macrophages bulk RNA-seq dataset
 files are available 1. Count Matrix (parada_et_al_2023_counts.csv): table
 with genes in rows and samples in columns. 2. Metadata
 (parada_et_al_2023_metadata.csv): table with Sample N°, Sample Name,
 experimental condition, and exp_date (experimental date) information. 3.
 Raw RNAseq Data in fastq.gz: 118 files corresponding to read 1 (R1) and
 read 2 (R2) from pair-end sequencing of 59 samples. Experimental
 conditions: * *uninfected*: macrophages isolated from control uninfected,
 untreated animals * *uninfected_3hk*: macrophages isolated from uninfected
 and 3hk treated animals * *exposed*: macrophages not containing bacteria
 isolated from salmonella infected animals * *exposed_3hk*: macrophages not
 containing bacteria isolated from salmonella infected and 3hk treated
 animals * *invaded*: Bacteria containing macrophages isolated from
 Salmonella infected animals. * *invaded_3hk*: Bacteria containing
 macrophages isolated from Salmonella infected and 3hk treated animals
 Experiment date information: * *10.16.2020*: Samples isolated from
 experiment conducted on 10/16/2020 * *10.29.2020*: Samples isolated from
 experiment conducted on 10/29/2020