title: GSM8072596: PCC 7806  Replicate C time 6; Microcystis aeruginosa; RNA-Seq
library_construction_protocol: RNA extraction was carried out using a Trizol reagent (Life Technologies) and the NucleoSpin miRNA kit (Macherey-Nagel). Firstly, 1 mL of Trizol preheated at 65 °C was added to the frozen pellets and incubated at 65 °C for 12 min with vortexing three times. After centrifugation (10 min at 4 °C and 12,000 rcf), the upper phase was transferred to a new tube and mixed with 500 µL of Trizol preheated at 65 °C. RNA extraction was achieved by adding 400 µL of chloroform, and incubating samples for 3 min at room temperature before centrifugation (10 min, 4°C, 12000 rcf). The upper phase was again transferred to a new tube for extraction with 400 µL of Trizol and 400 µL of chloroform with similar incubation and centrifugation conditions. The upper phase obtained from this step was then further processed using the NucleoSpin miRNA kit according to the manufacturer's instructions. The final purified RNA was eluted in 20 µL of RNase/DNase-free sterile water. The absence of genomic DNA contamination was confirmed by PCR targeting the 16S rRNA gene using specific primers. The quality and quantity of RNA were assessed using a Nanodrop spectrophotometer (ThermoFisher Scientific) and a Bioanalyzer (Agilent) with RNA Nano chips. Library preparation was conducted using the TruSeq Stranded mRNA sample preparation kit (Illumina, San Diego, California) following the manufacturer's instructions. Ribosomal RNA (rRNA) depletion was performed using the Ribo-Zero rRNA removal kit (bacteria, #MRZB12424, Illumina) with 5 µg of total RNA. The rRNA-depleted RNA was then fragmented using divalent ions at 94°C for 8 min. The resulting fragmented RNA samples were reverse-transcribed using random primers, followed by complementary-strand synthesis to generate double-stranded cDNA fragments. There was no need for an end repair step in this process. A 3'-end adenine was added, and specific Illumina adapters were ligated to the cDNA fragments. The ligated products were amplified by PCR. The quality of the resulting oriented libraries was assessed using Bioanalyzer DNA1000 Chips (Agilent, # 5067-1504) and quantified using spectrofluorimetry (Quant-iT™ High-Sensitivity DNA Assay Kit, #Q33120, Invitrogen). Sequencing was performed on the Illumina HiSeq2500 platform of the Institut Pasteur, generating single-end 65 bp reads with strand specificity. Each lane of sequencing accommodated a mixture of 18 multiplexed samples.