GSM5351268: P6_C1; Limnospira indica PCC 8005; RNA-Seq
Three replicates of 30 mL each of the retrieved irradiated cultures and the non-irradiated control cultures were immediately put on ice after gamma irradiation and centrifuged for 20 mins at 10,000 g and 4 °C, to collect the cell pellets in 15 mL conical FalconTM centrifuge tubes (BD Biosciences, Erembodegem, Belgium). The cell pellets were then transferred to 2 mL Eppendorf centrifuge tubes (Fisher Scientific) and as much as possible all Zarrouk medium was removed by additional centrifugation for 2 min at maximal speed. The pellets were washed three times with 1x Phosphate-Buffered Saline (PBS) and finally flash-frozen in liquid nitrogen until further analysis. RNA extraction was performed at 4oC using RiboPureTM- Bacteria Kit (Ambion-Life Technologies, Gent, Belgium), Direct-zolTM RNA miniPrep (Zymo Research, BaseClear Lab Products, Leiden, The Netherlands), RNA Clean and Concentrator-25 (Zymo Research) and the Turbo DNA-free kit (Ambion-Life Technologies, Gent, Belgium). The breakage of cell was performed by the RiboPureTM-Bacteria kit using Zirconia Beads in the lysis RNAwiz solution (both are kit components). The final volume of the lysis solution was adjusted according to the volume of the pellet. The released RNA was separated from cell debris by centrifugation at 10,000 g for 10 min at 4oC. The further purification of the released RNA was performed with the Direct-zolTM RNA Miniprep kit maintaining a 1:1 ratio of organic and aqueous phase, following the manufacturer's instructions. DNA was degraded with DNase 1 treatment (1 U/µL) and incubating at 37 °C for 30 mins. Obtained RNA was concentrated with the RNA Clean and Concentrator-25 kit. The quality and integrity of the RNA was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Diegem, Belgium). Library preparation was done using TruSeq Stranded Total RNA kit (Illumina, Brussels, Belgium) with fragmentation at 94°C for 3 minutes instead of 8 mins as to generate long fragments (an adaptation to the manual protocol for generation of long fragments). The amplification was performed using a PCR with 14 cycles. The quality check of the library was performed with an Agilent 2100 Bioanalyzer High Sensitivity Chip by means of qPCR. The amplicons were pooled together with size selection on a 2% E-GelTM Agarose Gel (ThermoFisher Scientific) and a final amplicon quality check was performed on the Agilent 2100 Bioanalyzer High Sensitivity Chip by means of qPCR. The final sequencing was performed on a HiSeq 3000 system (Illumina, San Diego, USA) with a single-read paired end preset at 150 bp (PE150 specifications).