title: RNA-Seq: Pseudomonas putida GB062: Substrate 10 mM glucose
design: Strains were grown to mid-exponential phase, cells were harvested by centrifugation at 4,500 rpm for 5 minutes. The cell pellets were flash frozen and sent to AZENTA Life Sciences for RNA library preparation and sequencing. RNA was quantified using a Qubit 2.0 Fluorometer (ThermoFisher Scientific) and integrity was checked using 4200 TapeStation (Agilent Technologies). The rRNA depletion sequencing libraries were prepared by using a QIAGEN FastSelect rRNA Bacteria Kit (Qiagen). RNA sequencing library preparation used NEBNext Ultra II RNA Library Preparation Kit for Illumina (NEB). Enriched RNAs were fragmented. First strand and second strand cDNA were synthesized. cDNA fragments were end repaired and adenylated and universal adapters were ligated to cDNA fragments, followed by index addition. Library enrichment was done with limited cycle PCR. The sequencing libraries were multiplexed and clustered on one lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000). The samples were sequenced using a 2x150 Paired End configuration. Raw sequence data (.bcl files) generated from Illumina HiSeq were converted into fastq files and de- multiplexed using the Illumina bcl2fastq program version 2.20.