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Whole transcriptome sequencing of HT22 immortalized mouse hippocampal cells treated with AA147 or veh for 16 h

ABSTRACT: Purpose: Define transcriptional remodeling associated with AA147 treatment (10uM) for 16 h in a mouse neuronal cell line. Methods: A minimum of 27M PE100 reads were generated for triplicates of vehicle vs. AA147 treated HT22 cells. Alignment of reads was performed using DNAstar Lasergene SeqManPro to the mouse genome GRCm39 assembly. Read counts generated by ArrayStar 12.2 QSeq package. Differential expression analysis performed using DESeq2. Results: We mapped between 27-32 M sequence reads per sample to the mouse genome and identified 16,473 transcripts with counts greater than 10. Of these, we identified 2,742 genes with significantly different expression between vehicle and AA147 treated cells. Conclusions: We observed substantial upregulation of prolactin and glutathione transferase genes, and upregulation of NRF2-associated pathway upregulation.

ORGANISM(S): Mus musculus

PROVIDER: GSE178964 | GEO | 2021/12/06

REPOSITORIES: GEO

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Project description:Purpose: T. gondii is an obligate intracellular parasite that remodels host transcriptome during infection. How T. gondii provokes these changes is not fully understood. In the present study we found that GRA18 is secreted in the infected host cell and associates with the β-catenin destruction complex to induce β-catenin levels, notably in the host nuclei. Therefore, we hypothesized that GRA18 could modulate the expression of β-catenin-dependent genes. To determine whether GRA18 induces changes in the host cell gene expression, we performed a transcriptomic analysis of BMDMs infected by Wild-Type and gra18 mutant strains of T. gondii. Methods: RNA profiles were generated from BMDMs (BALB/c mice) that were left uninfected or infected with the Pru ku80, Pru ku80 Δgra18, and Pru ku80 Δgra18, GRA18+++ strains (MOI = 6) for 18 hours and subsequently subjected or not to LPS stimulation for 4 hours.Total RNAs were extracted and purified using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy Plus Mini Kit (Qiagen). RNA-sequencing was performed by GENEWIZ (South Plainfield, NJ, USA). The RNA quality was checked with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California, USA) and Illumina TruSEQ RNA library prep and sequencing reagents were used following the manufacturer's recommendations (Illumina, San Diego, CA, USA). The samples were paired-end multiplex sequenced (2 × 125 bp) on the Illumina Hiseq 2500 platform and generated at least 70 million reads for each sample. The RNA-Seq reads (FASTQ) were processed and analyzed using the Lasergene Genomics Suite version 14 (DNASTAR, Madison, WI, USA) using default parameters. The paired-end reads were uploaded onto the SeqMan NGen (version 14, DNASTAR. Madison, WI, USA) platform for reference-based assembly using either the Mus musculus genome package (GRCm38.p3) or the Toxoplasma Type II ME49 strain (ToxoDB-24, ME49 genome) as reference template. The ArrayStar module (version 14, DNASTAR. Madison, WI, USA) was used for normalization, differential gene expression and statistical analysis of uniquely mapped paired-end reads using the default parameters. The expression data quantification and normalization were calculated using the RPKM (Reads Per Kilobase of transcript per Million mapped reads) normalization method. Conclusion: This approach identifies a specific set of genes that are regulated in a GRA18-dependent fashion in BMDM upon T. gondii infection. Particularly, GRA18 promotes the expression of chemokines Ccl17, Ccl22, and Ccl24.

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Whole transcriptome sequencing of HT22 immortalized mouse hippocampal cells treated with AA147 or veh for 16 h

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