Ga pipeline version 1.6

The TruSeq™ Stranded Total RNA Library Prep Kit from Illumina (San Diego, CA, USA) was used to construct the library for the experiment. After removing rRNA and adding fragmentation buffer, mRNA was randomly broken into small fragments of approximately 200 nt. Under the action of reverse transcriptase, one-strand cDNA was synthesized using random primers and mRNA as templates. For the second strand synthesis, dUTP was used instead of dTTP to form the base of the second strand of cDNA containing dTTP. Before PCR amplification, the second strand of cDNA was digested with the UNG enzyme so that only the first strand of cDNA was included in the library. Finally, Illumina Hiseq × 10 (2 × 150 bp read length) was used for sequencing. Processing of the original images to sequences, base-calling, and quality value calculations were performed using the Illumina GA Pipeline (version 1.6), in which 150 bp paired-end reads were obtained.

Total RNA was isolated from each of the samples using a Trizol Kit (Promega, USA), following the manufacturer's instructions. Then, the total RNA was treated with RNase-free DNase I (Takara Bio, Japan) to remove residual DNA. Poly (A) mRNA was isolated using oligo-dT beads (Qiagen). Fragmentation buffer was added to the mRNA to generate short fragments (200 nt). First-strand cDNA was synthesized by random hexamer-primed reverse transcription, and then second-strand cDNA was generated using RNase H and DNA polymerase I. The cDNA fragments were purified using a QIAquick PCR Extraction Kit. These purified fragments were then washed with EB buffer for end reparation poly (A) addition and ligated to sequencing adapters. Following agarose gel electrophoresis and extraction of cDNA from the gels, the cDNA fragments (200 ± 25 bp) were purified and enriched by PCR to construct the final cDNA library. The cDNA library was sequenced with an Illumina sequencing platform (Illumina HiSeq™2000) using paired-end technology. The processing of the original image to determine the sequences, in addition to the base calling and quality value calculations, were performed using Illumina GA Pipeline (version 1.6), from which 90 bp paired-end reads were obtained (Li et al., 2013 (link)).

For RNA-sequencing, total RNA was extracted from WT and the TfpNDM-hvKP strains grown to log phase in LB broth with shaking at 37 °C using TRIzol reagent (Invitrogen, Paisley, UK) and purified using the RNase-free DNaseI (TaKaRa, Dalian, China) following the manufacturer’s protocol. The concentration of purified RNA was quantified by the Qubit system (Thermo Fisher Scientific). RNA quality and quantity was evaluated on a 2100 Bio-analyzer (Agilent) using the AgilentRNA 6000 Pico Kit (Agilent Technologies, USA). The ribosomal RNA was removed from total RNA using the Ribo-Zero Magnetic Kit (Bacteria) (Epicentre Biotechnologies, Madison, Wisconsin, USA). cDNA libraries were produced by the Illumina TruSeq Stranded messenger RNA Sample Preparation Kit, and sequenced on the HiSeq 2000 system as 2 × 100-bp paired-end reads. Illumina GA Pipeline (version 1.6) was used to perform the original image process to sequences, base-calling and quality value calculation.