Illumina HiSeq 4000 paired end sequencing; RNA-seq of Synechococcus sp. WH8102 zur knockout mutant
RNA-seq of Synechococcus sp. WH8102 zur knockout mutant
Wild type Synechococcus sp. WH8102 and its zur knockout mutant were grown to the late-log phase of growth in non-Chelexed ASW-Zn and sub-cultured into ASW with different zinc content (0, 772 nM and 2.5 µM). Cultures were then grown to mid-exponential phase (OD750 ~0.3-0.4). Each condition had three biological replicates. Each culture was separated into sterile 50 mL Falcon tubes, centrifuged at 4000 rpm at 4 °C, separated from the supernatant by decantation and snap-frozen in liquid nitrogen. Cell pellets were thawed on ice in 1.5 mL of Z buffer (8 M guanidinium hydrochloride; 50 mM β-mercaptoethanol; 20 mM EDTA) at room temperature for 30 min. Two volumes of acidified phenol (pH 4.5) were added and mixtures were heated to 65˚C for 30 min, followed by the addition of a mixture of chloroform and isoamyl alcohol (24:1, v/v) for 15 min. The tubes were then centrifuged at 4000 rpm and the aqueous phase transferred to separate microcentrifuge tubes which were centrifuged again at 10,000 rpm and transferred into fresh tubes. RNA was precipitated by addition of 1 vol. of RNase-free isopropanol followed by incubation overnight at -20 ˚C. The tubes were then centrifuged at 13,000 rpm in a benchtop centrifuge at 4˚C for 30 mins. The pellets were then washed with 70% (v/v) RNase-free ethanol and further centrifuged for 15 min. Following decanting of the supernatant, pellets were dried at room temperature for 15 min and then re-suspended in 50 µL RNase-free water. DNA was removed using the TURBO DNA-freeTM kit (Ambion®/Life Technologies): double treatment for 30 minutes at 37 °C was applied with 2 units of DNase each time instead of one application with 1 unit. DNase was then inactivated according to the protocol and the samples were additionally purified using Zymo RNA Clean & Concentrator-5 using the manufacturer's instructions. The concentration of RNA and the purity of the samples were assessed using NanoDrop. The presence of DNA contamination was assessed by PCR with 16S rRNA gene primers (AGAGTTTGATCMTGGCTCAG and ACCTTGTTACGACTT). RNA integrity of the samples was assessed using an Agilent Bioanalyzer with an Agilent RNA 6000 Pico Kit following the manufacturer's instructions. Samples were re-purified using a Qiagen RNeasy Kit. RNA samples were sent to the Centre for Genomic Research, Institute of Integrative Biology, at the University of Liverpool for library preparation and sequencing. A RiboZero kit, Illumina was then used for rRNA depletion and a NEB Next Ultra Directional RNA library preparation kit used for preparation of dual-indexed, strand-specific RNA-seq libraries.