RNA Extraction and Transcriptome Analysis of E. coli

Total RNA was isolated from E. coli strains that were grown for 48 hr with 0.6 g/L malate as carbon sources and H₂ supply. Total RNA was extracted using Trizol^® Reagent (Invitrogen, Carlsbad, CA, USA) according to the instruction manual. Purified RNA was quantified at an optical density (O.D.) of 260 nm using a ND-1000 spectrophotometer (Nanodrop Technology, Waltham, MA, USA) and qualitated using a Bioanalyzer 2100 (Agilent Technology, Santa Clara, CA, USA) with the RNA 6000 LabChip kit (Agilent Technology, Wilmington, DE, USA). All RNA sample preparation procedures were carried out according to the Illumina’s official protocol. The SureSelect XT HS2 mRNA Library Preparation kit (Agilent, Santa Clara, CA, USA) was used for library construction, followed by AMPure XP beads’ (Beckman Coulter, Brea, CA, USA) size selection. The sequence was determined using Illumina’s sequencing-by-synthesis (SBS) technology (Illumina, San Diego, CA, USA). Sequencing data (FASTQ reads) were generated using Welgene Biotech’s pipeline based on Illumina’s basecalling program bcl2fastq v2.20. Differential expression analysis was performed using StringTie (StringTir v2.1.4) and DEseq (DEseq v1.39.0) or DEseq2 (DEseq2 v1.28.1) with genome bias detection/correction and Welgene Biotech’s in-house pipeline.