RNA-Seq of Acetate-R2: Methanosarcina barkeri
Cells representing the mid-exponential phase were collected from all treatments for total RNA extraction, which was performed using a RNAprep pure Cell/Bacteria Kit (Cat. No. DP430; TIANGEN, Beijing, China). RNA purity and concentrations in the samples were determined by gel electrophoresis and a NanoDrop2000 spectrophotometer (Thermo Scientific, Waltham, MA). Ribosomal RNA was removed from total RNA using the RiboMinus TM kit (Lot. No.1539791, Invitrogen, Waltham, MA). Samples were then submitted to GENEWIZ (Suzhou, China) for library construction and sequencing on an Illumina HiSeq 4000 system (Illumina, San Diego, CA). The sequencing reads were mapped to the M. barkeri DSM800 genome using Bowtie 2. HTSeq 2.0 was used to determine the read numbers mapped to each gene, after which the FPKM (fragments per kilobase per million reads) for each gene could be calculated. Differential transcription analysis for the substrate conditions (acetate vs. CO2/H2, acetate vs. methanol, and methanol vs. CO2/H2) was performed using the DESeq2 (1.42.0) R package. Genes with an adjusted p-value < 0.01 from the DESeq2 package and a FPKM value >100 in the groups sample were classified as demonstrating differential transcription.