Illumina HiSeq 3000 sequencing; Transcriptomic response analysis of Escherichia coli to palladium stress
When the OD600 reached 0.3, 4 biological replicates were challenged with 100 Î¼M Pd2+ in the form of sodium tetrachloropalladate (II) dissolved in 0.01 M nitric acid (similar amounts of Milli-Q water were added to the 4 control replicates). The cultures were incubated at 37oC with shaking at 200 rpm for 10 minutes. The cells were harvested by centrifuging at 4200 x g for 10 minutes at 4oC. Then, the pellets were resuspended in 1 ml of 20 mM MOPS buffer (pH 7) and transferred to 2 ml Eppendorf tubes to remove excess heavy metal ions that might impact the subsequent RNA isolation and analysis. The tubes were centrifuged at 6000 x g for 2 minutes at 4oC. The supernatant was removed, and the tubes were immediately dipped into liquid nitrogen, stored at -80oC, and later shipped on dry ice.  RNA isolation was done by RNeasy kit (Qiagen) The mRNA was fragmented and random hexamer primers were used for cDNA synthesis. Adapter ligation and adapter-specific PCR amplification were used to generate libraries of 150 bp reads. More than 10 million reads were generated per sample. The reads were paired-end sequenced by an Illumina sequencing platform.