GSM9149809: Zymomonas mobilis WT Aerobic RNA-seq Replicate A; Zymomonas mobilis subsp. mobilis ZM4 = ATCC 31821; RNA-Seq
The RNA was extracted, the same day as the cells were harvest, following standard hot phenol:chlorofom protocol, and ethanol precipitated overnight at -80º C. The RNA then was resuspended in RNase free water and treated with DNAse I at 37ºC for 1 hour Then the RNA was reextracted and precipitated as before. The clean RNA was resuspended in RNAse free water. Samples were initially treated with TURBO DNase (Thermo Fisher Scientific, Waltham, MA, USA) to remove DNA contaminants. Strand-specific RNA sequencing libraries was prepared by using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, the enriched RNAs were fragmented for 8 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. The second strand of cDNA was marked by incorporating dUTP during the synthesis. cDNA fragments were adenylated at 3'ends, and indexed adapter was ligated to cDNA fragments. Limited cycle PCR was used for library enrichment. The incorporated dUTP in second strand cDNA quenched the amplification of second strand, which helped to preserve the strand specificity. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).  The sequencing libraries were clustered on the flowcell. After clustering, the flowcell was loaded on the Illumina instrument (4000 or equivalent) according to manufacturer's instructions.  The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the Control software. Raw sequence data (.bcl files) generated the sequencer were converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.20 software. One mismatch was allowed for index sequence identification.