GSM8654266: delta tsdA delta shdrR cells grown on methanol and thiosulfate (biol rep 2); Hyphomicrobium denitrificans; ssRNA-seq
From the frozen pellets, the RNA was purified with the FastGene RNA Premium Kit (NIPPON Genetics EUROPE, Düren, Germany) according to the manufacturer's instructions. A modification was introduced regarding the cell lysis step. After addition of the lysis buffer that contained 1% (v/v) 2-mercaptoethanol, cells were disrupted by bead beating (Bead Ruptor 12 Bead Mill Homogenizer, Omni International, Kennesaw, GA, USA) for three cycles of 30 s at maximum speed and incubation on ice for 1 min. RNA quality was checked on 1% agarose gels and its concentration was measured using NanoPhotometer NP80 (IMPLEN, Munich, Germany). The RNA was shipped on dry ice to Eurofins Genomics GmbH (Ebersberg, Germany). The subsequent analysis pipeline included rRNA depletion, library preparation (mRNA fragmentation, strand specific cDNA synthesis), Illumina paired end sequencing (2 x 150 bp, minimum 10 MB reads)), and bioinformatic analysis (mapping against the reference genome, identification and quantification of transcripts, pairwise comparison of expression levels and determination of significant fold differences) and was conducted by the company. Strand-specific cDNA library. RNA-seq Technology: Genome Sequencer Illumina NovaSeq. Sequence mode: NovaSeq 6000 S4 PE150 XP