Ribo zero rrna removal kit for bacteria

Approximately 1.3 μg of total RNA for each replicate sample was ribodepleted using the Ribo-Zero rRNA removal kit for bacteria (Epicentre) according to the manufacturer’s protocol. The TruSeq total RNA stranded kit (Illumina) was used for the preparation of libraries. A Quibit spectrophotometer measured their quantity and a Tapestation on a DNA High-sensitivity chip (Agilent Technologies) determined their quality. The 12 generated libraries were pooled at equimolarity and loaded at 7 pM for clustering. An Illumina HiSeq 4000 instrument was used to sequence 100-bp single reads per sample (iGE3 Genomics platform of the University of Geneva). FastQC v.0.11.5 was used for sequencing quality control and reads were mapped to the NCBI NC_008463.1Pseudomonas aeruginosa UCBP-PA14 reference genome using BWA aligner v.0.7.17 software. The counts were normalized according to the relative number of reads for each library, and genes having a count above 1 count per million reads (cpm) in at least 3 samples were kept for the analysis. The starting raw gene number of the set was 6,100. The final filtered data set consisted of 6,043 genes after excluding poorly or nonexpressed genes.

For each sample, 20 µl total RNA was added to an RNAstable tube (Biomatrica), gently mixed with the preservation agent and left to air-dry in a biosafety cabinet for 48 h. Samples were shipped at ambient temperature to Macrogen (Geumcheon-gu, Seoul, Republic of Korea) for RNA-seq. Ribosomal RNA was removed by treatment with the Ribo-Zero rRNA removal kit for bacteria (Epicentre), followed by 100 bp paired-end, stranded library construction using the TruSeq rapid SBS Kit (Illumina). Libraries were sequenced on either the HiSeq2000 or the HiSeq2500 platform (Illumina). All samples were extracted from two separate experiments to account for biological variation, except for MSHR8442, which was extracted thrice. Between 36 and 80 million reads were generated for each sequence, corresponding to between 3.6 and 8.1 billion bp each.

Total RNA quality was assessed using an Agilent 2100 bioanalyzer and RNA Nano Chip kit (Agilent Technologies) to ensure that the RNA integrity number (RIN) was ≥7 and that there were no RIN value outliers. rRNA was removed from 1 μg of RNA with a Ribo-Zero rRNA removal kit for bacteria (Epicentre Biotechnologies). To generate a directional signal in the RNA-Seq data, libraries were constructed from first-strand cDNA using the ScriptSeq v2 RNA-Seq library preparation kit (Epicentre Biotechnologies). Briefly, 50 ng of rRNA-depleted RNA was fragmented and reverse transcribed using random primers containing a 5ʹ tagging sequence, followed by 3ʹ-end tagging with a terminus-tagging oligonucleotide to yield di-tagged single-stranded cDNA. Following purification by a magnetic bead-based approach, the di-tagged cDNA was amplified by limit-cycle PCR using primer pairs that anneal to tagging sequences and add adaptor sequences required for sequencing cluster generation. Amplified RNA-Seq libraries were purified using an AMPure XP System (Beckman Coulter). The quality of libraries was determined via an Agilent 2200 TapeStation using high sensitivity D1000 tape and quantified using a Kapa SYBR Fast qPCR kit (KAPA Biosystems, Inc). One hundred fifty-base pair sequence reads were generated using the Illumina HiSeq 4000 platform.