GSM9352804: SE50/110 ΔtreY, acarbose fermentation, 56 hr, rep 1; Actinoplanes; RNA-Seq
Total RNA was isolated using the RNeasy mini kit (Qiagen, Germany), after which the RNA concentration and quality were determined by the Qubit®3.0 Fluorometer (Life Technologies, USA) and the Nanodrop One spectrophotometer (Thermo Fisher Scientific Inc., USA). Integrity of total RNA was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., USA), and samples with RNA integrity number (RIN) values above 7.0 were used for sequencing. One micrograms of RNA were used as input material for the RNA sample preparations. RNA-seq strand-specific libraries were constructed using the VAHTS Total RNA-seq (H/M/R) Library Prep Kit (Vazyme, China) according to the manufacturer's instructions. Briefly, RNA was purified by magnetic beads after removal of rRNA. Then RNA was fragmented into small pieces using divalent cations for 8 min at 94°C. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. These cDNA fragments then go through the end repair process, addition of a single 'A' base, and ligation of the adapters. The products were purified and enriched by PCR to create the final cDNA library. Purified libraries were quantified and validated by Qubit® 3.0 Fluorometer and Agilent 2100 bioanalyzer to confirm the insert size and calculate the mole concentration. Cluster was generated by cBot after the library diluted to 10 pM and then were sequenced on the Illumina NovaSeq 6000 platform (Illumina, USA). The library construction and sequencing were performed by Sinotech Genomics Co., Ltd (Shanghai, China).