RNA-sequencing Kombucha fermentation
For the RNA extraction procedure, fermentations were carried out as described above and 1 mL of kombucha supernatant was collected after 24 h of fermentation. Cells were centrifuged at 5,000 rpm for 3 min and the supernatant was discarded. Then, the pellet was washed twice with 50 mM EDTA and centrifuged at 5,000 rpm for 3 min. For the cell wall digestion, 1 mL of Y1 solution (Qiagen Handbook) + 14 L of beta-mercaptoethanol + Zymolyase (40 U) was added. RNA was extracted using the E.Z.N.A Total RNA Kit I (OMEGA) according to the manufacturers instructions and treated with DNAse I (Promega) to remove genomic DNA traces. RNA was cleaned according to the GeneJET RNA Cleanup and Concentration Micro Kit. The purified RNA was quantified by Qubit using the Qubit RNA BR Assay Kits and the integrity was determined with the TapeStation kit using the RNA ScreenTape Analysis. Finally, cDNA libraries were prepared using TruSeq Stranded Total RNA kit (Illumina) and sequenced on an Illumina NextSeq500 platform by high-throughput paired-end sequencing