GSM5946204: GB062_gfg_2; Pseudomonas putida KT2440; RNA-Seq
Cells pelleted from 15 mL of M9 minimal medium were flash frozen and stored at −80°C. Cells were resuspended in TRIzol (ThermoFisher-Invitrogen) and processed according to the manufacturer's protocol. The samples were then purified on an RNeasy column (Qiagen) with on-column DNase digestion. RNA was eluted off the column in 35 μL RNase free H2O (Qiagen). The RNA concentration was quantified using a Nanodrop 1000 (ThermoScientific) and RNA quality was verified by obtaining RNA Integrity Numbers (RIN) using an RNA 6000 Nanochip on an Agilent 2100 Bioanalyzer (Agilent). Ribosomal RNA was depleted using a RiboZero rRNA Removal Kit for bacteria (Illumina). The depleted sample was purified on an RNA Clean & Concentrator-5 (Zymo Research), and then the depleted material was quantified using a Nanodrop 1000 and visualized on an Agilent 2100 Bioanalyzer instrument with an RNA 6000 Nanochip (Agilent). RNA depleted of ribosomal RNA was used as input material to synthesize cDNA libraries using a ScriptSeq v2 RNA-Seq Library Preparation Kit (Illumina) and TruSeq compatible barcodes. Pooled barcoded libraries were sequenced in one direction for 75 bases (SE75) on an Illumina NextSeq500 (Illumina) and de-multiplexed as a sequencing service provided by Vanderbilt 2.9 (VUMC VANTAGE Vanderbilt University Medical Center Technologies for Advanced Genomics).