Indexed adapters

Genomic DNA was isolated from multiple sources including tissue, blood, respiratory samples, urine and water (environmental), and was quantified using a Qubit fluorometer (Life Technologies, USA). The quality and quantity of DNA were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, MA, USA) and PFGE gel electrophoresis, respectively. The qualified DNA was cut into fragments by restriction enzymes. DNA fragments were end-repaired using deoxyadenosine bases before ligation with Illumina indexed adapters, amplified for 10 cycles of PCR, and sequenced employing v2 and v3 chemistry with paired-end 2 × 151 bp reads on NovaSeq 6000 (Illumina, USA). Output data files were de-multiplexed and transformed with Casava v.1.8.2. into FASTQ files (Illumina, Inc, USA).

A total of 18 samples (three conditions, six biological replicates per condition) were sequenced for this study. Two micrograms of total RNA was used to build sequencing libraries using Illumina TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA). Briefly, mRNAs were purified using poly-T magnetic beads then chemically fragmented (around 300 bp) under 94 °C. First and second strand cDNA synthesis were consecutively performed according to the manufacturer’s protocol. After that, cDNA fragments were adenylated on 3′ ends, and Illumina indexed adapters were ligated on both ends. Specific tagged cDNA fragments were then enriched in a PCR program including a denaturation step at 98 °C (30 s) followed by 10 cycles of amplification (98 °C for 10 s, 60 °C for 30 s and 72 °C for 30 s) and a final 30 s step at 72 °C. Finally, pooled libraries were sequenced on one lane of Illumina HiSeq 3000 platform, resulting in 150 pb paired-end reads. Transcriptomic data and experimental details are available in NCBI’s Gene Expression Omnibus [32 (link)] and are accessible through GEO Series accession number GSE189211 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE189211).

Genomic DNA isolated from freshly grown overnight culture (QIAamp DNA minikit; Qiagen, Germany) was quantified using Qubit fluorometer (Life Technologies, USA). Sequencing library preparation was by Illumina Nextera XT DNA sample preparation kit (Illumina, USA). Genomic DNA samples were fragmented using Covaris M-series (M220) at temperature of 5.5 to 6 °C for 40 seconds. DNA fragments were end repaired using dA bases before ligation with Illumina indexed adapters, amplified for 10 cycles of PCR and sequenced employing v2 and v3 chemistry with paired-end 2 × 151 bp reads on Illumina MiSeq (Illumina, USA). Output data files were de-multiplexed and transformed with Casava v.1.8.2. into FASTQ files (Illumina, Inc, USA).