Effects of ppGpp on gene expression during nitrogen deprivation in Arabidopsis.. G4PLAST_1

RNA-seq experiments on Arabidopsis thaliana wildtype (Col-0) and OX:RSH1 (overexpression of RSH1, low ppGpp levels) seedlings. RNA sequencing was performed with three biological replicates on 100 bulked seedlings per line grown on +N medium for eight days or -N medium for 12 days. RNA was extracted from frozen seedling powder with Nucleozol (Macherey-Nagel) with 4-bromoanisole to reduce DNA and anthocyanin contamination. Total RNA was cleaned and concentrated using RNA Clean & ConcentratorTM-25 (Zymo Research) according to the manufacturer's instructions Genomic DNA was removed by treatment with DNase. RNA-seq libraries were constructed by the POPS platform (IPS2) using the TruSeq Stranded mRNA library prep kit (Illumina) with RiboZero plant (Illumina). Libraries were sequenced in single-end (SE) with a read length of 75 bases for each read on a NextSeq500 (Illumina). Approximately 30 million reads by sample were generated. Adapter sequences and bases with a Q-Score below 20 were trimmed out from reads using Trimmomatic (v0.36) (Bolger et al., 2014) and reads shorter than 30 bases after trimming were discarded. Reads corresponding to rRNA sequences were removed using sortMeRNA (v2.1)(Kopylova et al., 2012) against the silva-bac-16s-id90, silva-bac-23s-id98, silva-euk-18s-id95 and silva-euk-28s-id98 databases. Read quality checks were performed using FastQC (Version 0.11.5)(Andrews, 2010).