RNA-seq of Saccharolobus islandicus synchronized culture - sample 2h30_11_S24_R1_001
Cells were plated on solid media and 15 single colonies were inoculated in liquid media. forming 15 cultures that represent completely independent biological replicates. The 15 biological replicates were grown in 3 different batches (5 cultures per batch), each batch was grown and synchronized at a different time (5 cultures at a time). RNA was then isolated from each batch on different days, no variance between batches was found using principal component analysis (see supplementary table S1 for batch number for each sample). The total RNA was extracted from the 15 biological replicates at three different time-points after synchronization: 2h30, 4h and 6h (Fig. 1B). The time points were selected based on the results of the flow cytometry analysis which showed that samples at these three time points are enriched in cells in the M-G1, S and G2 phases, respectively (Figure 1C). Total RNA was extracted using TRI Reagent (SIGMA-Aldrich), following the manufacturers protocol, and treated with DNase (TURBO DNA-free kit; Invitrogen) following the manufacturers instructions. DNase treated samples were further purified with RNeasy Mini Kit (Qiagen). RNA samples were quantified using a Qubit Fluorometer (Thermo Fischer Scientific) and assessed for quality with a BioAnalyzer (Agilent) (see supplementary table S1 for quality control parameters for each sample).  Libraries were prepared using the Illumina Stranded Total RNA Prep, Ligation with Illumina Ribo-Zero Plus kit, with custom ribodepletion. Following PCR amplification, all samples underwent two rounds of purification with AMPure beads (Beckman Coulter) to remove small fragments. Libraries were subsequently validated using both the Qubit Fluorometer and the Fragment Analyzer (Agilent). Sequencing was performed on an Illumina NextSeq 2000 sequencer with a P3 50-cycle kit and a target of 30 to 40 million reads per sample. The reads obtained were mapped to the reference S. islandicus REY15A genome (RefSeq accession number: NC_017276) with Bowtie-290 using default parameters.