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Transcriptional Regulation of Nitrogen and Nitrogen-Related Metabolism in Arabidopsis

ABSTRACT: Purpose: Investigate transcriptional changes by genetic perturbation of specific transcription factors under sufficient and limiting nitrate conditions. Methods: 9 day old roots were harvested between 1-2pm (get hours after “sunrise”) and frozen in liquid N2. RNA-seq libraries were prepared following the BRAD-Seq DGE protocol (four biological replicates per genotype and treatment). Libraries were sequenced using the Ilummina HiSeq 3000 in SR50 mode. Two technical replicates libraries were created from each RNA sample; after assessing sufficient reproducibility, counts across technical replicates were pooled together. Reads were processed with fastx_trimmer for barcode removal (-f 9 -v -Q 33) and trimmed by quality with reaper (paremeters: -geom no-bc -dust-suffix-late 10/ACTG -dust-suffix 10/ACTG --noqc -nnn-check 1/1 -qqq-check 33/10 -clean-length 30 -tri 20 -polya 5 --bcq-late), then mapped to the reference genome of Arabidopsis thaliana (TAIR v10) using bowtie (-a --best --strata -n 1 -m 1 -p 4 --sam --tryhard) with subsequent conversion to BAM format using samtools and counts were obtained with HTSeq-count.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE107988 | GEO | 2018/07/30

REPOSITORIES: GEO

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Project description:Aim: We generated mice lacking Irs2 which mediates signaling pathways from insulin and IL-4 specifically in lyzM Cre expressing cells. We isolated BMDM from control and Irs2LyzM-/- mice and compared their differential gene expression under veihicle, IL-4 and LPS treatment by performing mRNA sequencing studies. Methods: BMDM from control and Irs2LyzM-/- mice were treated for 24 hours with vehicle, IL-4 (10 ng/ml) or LPS (100 ng/ml). RNA integrity was confirmed and concentration measured using a Bioanalyzer. cDNA libraries were created from up to 5 μg total RNA. There were three (Irs2LyzM-/-) and four (control) biological replicates for each group and each biological replicate was divided into eight technical replicates and sequenced on individual lanes on the Illumina HiSeq 2000 (100 bp paired-end read length) at the Sanger Institute. Raw reads from the sequenced RNAseq libraries were mapped with Tophat splice junction aligner (Kim, Pertea et al. 2013), version 2.0.8 against Ensembl mouse genome reference sequence assembly (mm9) and transcript annotations. All parameters were set to default except inner distance between mate pairs (r = 130). Gene-based read counts were then obtained using the HTSeq count module (version 0.5.4p3). Differential expression analysis was performed on the counts data using the DESeq2 Bioconductor package (version 1.16.1). The GSEA preranked tool from GSEA version 2.2.4 was used forGeneset enrichment analysis. Gene ontology enrichment analysis was also performed using the topGo Bioconductor package separately for up and down-regulated genes in the comparisons analysed ( A. Alexa, J. Rahnenfuhrer, topGO: Enrichment Analysis for Gene Ontology. R package version 2.24.0 (2016)). Results: The mRNA seq. results reveal an anti- inflammatory transcriptional profile in LPS-treated Irs2LyzM-/- BMDM compared to control BMDM. Deletion of Irs2 in BMDM under all conditions (vehicle, IL-4 and LPS) manifest alteratons in genes involved in scavenging catecholamines and supporting increased sympathetic innervation.

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Transcriptional Regulation of Nitrogen and Nitrogen-Related Metabolism in Arabidopsis

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