Illumina HiSeq 4000 sequencing; RNA-seq comparison of Clostridium acetobutylicum wild-type and pks mutant under batch fermentation conditions.
RNA-seq comparison of Clostridium acetobutylicum wild-type and pks mutant under batch fermentation conditions.
Samples (10 mL) of fermentation broth were taken in biological triplicate from bioreactor fermentations of wild-type and ∆pks C. acetobutylicum 26 hours post-inoculation. P2 liquid media (80 g/L initial glucose) was used. The samples were centrifuged (4,000 xg, 10 min, 4oC) and the pellets were resuspended and stored in RNAprotect Cell Reagent (Qiagen).  We performed a targeted in-frame deletion of the pks gene (ca_c3355) using an allelic exchange method previously developed forC. acetobutylicum15. The resulting mutant (Δpks) was confirmed by PCR analysis  Total RNA was extracted using an RNeasy Mini kit (Qiagen) according the manufacturer’s instructions. An on-column DNase treatment was performed using DNase I (RNase-free) (NEB).  RNA quality control, library construction, and library sequencing were performed by the University of California-Berkeley QB3 Functional Genomics Laboratory and Vincent J. Coates Genomic Sequencing Laboratory. RNA quality and concentration was assessed using a nanochip on an Agilent 2100 Bioanalyzer. Bacterial 16S and 23S rRNA was removed using a RiboZero kit (Illumina). The resulting mRNA was converted to an RNA-Seq library using an Apollo 324 with PrepX RNAseq Library Prep Kits (WaferGen Biosystems, Fremont, CA). RNA library sequencing was performed on an Illumina HiSeq4000 with 50 bp single end reads. Sequencing reads (50 bp) were processed and mapped to the C. acetobutylicum ATCC 824 genome (NCBI accession NC_003030.1 [chromosome] and NC_001988.2 [megaplasmid]) using CLC Genomics Workbench 9.0 with default parameters. Reads that did not uniquely align to the genome were discarded. Differences in gene expression between wild-type and ∆pks C. acetobutylicum were calculated using the same software. Genes were considered differentially expressed with p-value < 0.003 and | normalized fold change | > 2.0. Results of the RNA-Seq analysis are presented in Supplementary Table 3. STRING analysis was performed to determine putative protein-protein interactions between the differentially expressed genes revealed by RNA-Seq analysis.RNA quality and concentration was assessed using a nanochip on an Agilent 2100 Bioanalyzer. Bacterial 16S and 23S rRNA was removed using a RiboZero kit (Illumina). The resulting mRNA was converted to an RNA-Seq library using an mRNA-Seq library construction kit (Illumina).