GSM2978360: PDD_P2_01; Escherichia coli BW25113; RNA-Seq
after treament bacteria were resuspended in QiaGen RNAprotect Bacteria Reagent (QiaGen #76506), incubated for 5min, centrifuged, and flash frozen on dry ice. Total RNA was extracted by incubating bacteria in Enzymatic Lysis Buffer  (lysozyme & proteinase K) for 5 min followed by addition of QiaGen RLT Lysis Buffer and RNA purification  using the QiaGen RNeasy Mini kit combined with DNase treatment on a solid support (QiaGen #74104). RNA quality assessment and quantification was performed using microfluidic chip analysis on an Agilent 2100 bioanalyzer (Agilent Technologies). For RNA-sequencing library preparation, 1000 ng total RNA was used as input. First, bacterial ribosomal RNA was depleted using the Ribo-Zero Magnetic Kit Bacteria (Illumina #MRZB12424). After depletion, RNA was resuspended in TruSeq Total RNA Sample Prep Kit Fragmentation buffer (8.5 ul RNA and 8.5 buffer) and reversed transcribed into cDNA using random hexamer primer. Then cDNA was further processed for the construction of sequencing libraries according to the manufacturer's recommendations using the TruSeq Stranded mRNA Sample Prep Kit (Illimina #RS-122-2101). Sequencing was performed with the Illumina TruSeq SBS Kit v4-HS chemistry (Illumina #FC-401-4003) on an Illumina HiSeq2500 instrument with 50 cycles of 2x50 bp paired-end sequencing.