RNAseq of Acinetobacter baumannii 5116 stimulated by meropenem
Firstly, the rRNA was depleted from 1 microgram of total RNA using illumina MRZB12424 Ribo-Zero rRNA Removal Kit (Bacteria) (Illumina, San Diego, CA, USA ). Then, the first-strand cDNA was synthesized using ProtoScript  Reverse Transcriptase (New England BioLabs, Ipswich, MA, USA) at 25 C for 10 min; 42 C for 15 min; 70 C for 15 min. The second-strand cDNA was synthesized using NEBNext Second Strand Synthesis Reaction Buffer and dATP, dGTP, dCTP, dUTP mix  (New England BioLabs, Ipswich, MA, USA) at 16 C for 1 h. Resulted cDNA was purified with Agencourt AMPure XP beads (Beckman Coulter, Brea, CA) and end repaired with NEBNext End Repair Reaction Buffer and Enzyme Mix (New England BioLabs, Ipswich, MA, USA) at 20 C for 30 min; 65 C for 30 min. Sequencing adapters were ligated using NEBNext Adaptor for Illumina (New England BioLabs, Ipswich, MA, USA) at 20 C for 15 min. The second-strand cDNA was then degraded using the USER enzyme mix (New England BioLabs, Ipswich, MA, USA) at 37 C for 15 min and the product was purified by Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). Finally, the clustering of the index-coded samples was performed on a cBot Cluster Generation System using NEBNext Q5 Hot Start HiFi PCR Master Mix(New England Biolabs, Ipswich, MA, USA). After cluster generation, sequencing was performed using the Illumina X ten platform with pair-end 150 base reads.