Ribo zero plus rrna depletion kit

Bacteria were harvested by centrifugation (15,000 g for 1 min, 25°C). Total RNA was extracted using a Presto mini RNA bacteria kit according to the manufacturer’s instructions. The concentration of each extracted RNA sample was determined by using a Qubit fluorometer. The purity and integrity were assessed using a NanoPhotometer and an Agilent FA5200, respectively. For the RNA sequencing, cDNA libraries were prepared using the Illumina Ribo-Zero Plus rRNA Depletion kit according to the manufacturer’s instructions. Illumina pair-end reads (150-bp) RNA-seq were performed on an MiSeq platform by HGT Co. (Taiwan). The reads were trimmed and filtered for data quality control using a Phred score of 30. RNA-seq reads were mapped against the V. dispar ATCC 17748^T reference genome. The raw counts were normalized using the trimmed mean of M values (TMM) method. The normalized gene matrix was analyzed using iDEP (45 (link)), a website providing integrated RNA-seq data analysis applications. Differential expressed genes analysis, principal-component analysis (PCA), and hierarchical clustering were performed using this web application. Hierarchical clustering was carried out with the average linkage method by correlation distance. KEGG enrichment pathway analysis was performed with KOBAS 2.0 (46 (link)).

Transcriptomes were generated for six timepoints from each condition and RNA was extracted from frozen pellets using Qiagen RNEasy Mini Kit, total RNA was quantified and quality-assessed using the 2100 Expert Prokaryote Total RNA Pico kit and Agilent Bioanalyzer, rRNA sequences were depleted using an Illumina Ribo-Zero Plus rRNA Depletion Kit, libraries were created using the Illumina TruSeq Stranded Total RNA Kit, and sequenced on a HiSeq-2500 1 TB device, yielding paired-end 2 × 101 bp reads. Between 13 and 29 million reads were obtained per sample (median = 18.5 million reads). BBtools v35.84 following JGI’s default pipeline was used on raw reads to remove those containing two or more “N” bases with an average quality score across the read <10, with a length ≤51 bp, containing known Illumina artifacts, or mapping to PhiX, human, cat, dog, and mouse genomes with the identity of ≥93%. BBtools v35.84 trimmed reads to remove known Illumina artifacts in 5’ and 3’ ends, or when with a base quality score under 6 on 3’. Differential expression analyses were conducted with a custom-made R script used in numerous prior phage-host ‘omics studies [30 , 42 (link), 43 (link)]. Treatment samples were compared to their corresponding untreated control at each time point (e.g., T2 cyanovirocells versus T2 cyanobacteria only; T2 cyanovirocells with protist versus T2 cyanobacteria only).