Miniseq system denature and dilute libraries guide

Amplicons from the second round of PCR amplification were subjected to size selection (AMPure XP paramagnetic beads, Beckman Coulter, Brea, CA, USA), followed by concentration determination. The concentration of each subject library was measured using an ultrasensitive fluorescent nucleic acid dye that allowed quantification of double-stranded DNA (PicoGreen dsDNA quantification reagent, Thermo Fisher, Waltham, MA, USA). The final library was prepared by equimolar pooling of the individual subject libraries to a final library with a molar concentration of 10 nM. The final library was diluted and denatured according to the recommendations in the Illumina MiniSeq System Denature and Dilute Libraries Guide. The final library was sequenced on the Illumina MiniSeq sequencer using the MiniSeq Mid Output Kit (300 cycles) with 150 bp paired-end reads.

Mammalian RNA-depleted samples (n = 3 per condition) were directly used to deplete bacterial rRNA and cDNA libraries construction following Ribo-zero (Illumina) manufacturer’s recommendations. Successful rRNA depletion was verified on a Bioanalyzer using the DNA high-sensitivity kit (Agilent), and the final concentrations were determined using the Qubit 4 Fluorometer (Invitrogen).
Samples were processed immediately, according to Illumina Stranded Total RNA Prep with Ribo-zero Plus TruSeq Stranded mRNA Library Prep recommendations. In the end, cDNA libraries were evaluated on a Bioanalyzer (Agilent) to confirm average fragments between the range of 200–300 bp, and final concentrations were measured on a Qubit. The libraries’ normalization and pooling were performed according to the MiniSeq System Denature and Dilute Libraries Guide: protocol A from Illumina. For each run, a library of three samples with equimolar amounts was combined with PhiX control (spike-in of 0.5–2%) and paired-end sequenced (2 × 75bp) on an in-house Illumina Miniseq sequencer. After the first sequencing run, specific samples were re-sequenced to increase the sequencing depth of the phage and bacterial transcriptomes and improve the statistical power to detect differentially expressed genes.

The fungal ITS1 region from each sample was amplified with the primer pair ITS1‐F/ITS2 that contain in addition to the primer sequence the Illumina adaptors and a unique barcode sequence (Walters et al., 2016). For each sample, three technical replicates were prepared with the epMotion 5070 robotic pipetting system (Eppendorf). Reactions were set up in 20 μl volumes, containing 10 μl Sso Advanced Universal SYBR Green Supermix (Bio‐Rad Laboratories), 6.8 μl Nuclease‐free H₂O, 0.4 μl primers (10 μM, biomers.net), 0.4 μl MgCl₂ and 2 μl template. Cycling conditions comprised 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, 51 °C for 30 s and 72 °C for 1 min 30 s. ITS1 amplicons were purified with QIAquick Gel Extraction Kit (Qiagen) and quantified with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). Purified PCR amplicons containing unique barcodes from each sample were diluted to 1 nM solutions and pooled. Library preparation was carried out according to the MiniSeq System Denature and Dilute Libraries Guide from Illumina. Sequencing was performed on the Illumina MiniSeq as described previously for 16S rRNA genes (Pichler et al., 2018), containing 350 μl library (1.8 pM), 150 μl denatured genome library (1.8 pM) and 15 μl denatured PhiX control, and the Illumina MiniSeq Mid Output Kit (300‐cycles).