Transcriptional Regulation of Nitrogen and Nitrogen-Related Metabolism in Arabidopsis. Transcriptional Regulation of Nitrogen and Nitrogen-Related Metabolism in Arabidopsis

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. Overall design: Wild-type and 12 T-DNA insertion lines were grown in media with 1 or 10 mM KNO3. mRNA from 9 days old roots was collected for RNA-seq libraries using BRAD-seq.