title: 16S rRNA seq of cold-pressed oil
design: Whole  DNA from oil samples was extracted using the DNeasy Blood and Tissue Kit (Qiagen GmbH, Hilden, Germany) in accordance to the manufacturers protocol for Gram-negative bacteria with some siginificant changes due to the nature of the material. For this purpose 50 ml of each sample was centrifugated  (9,000 g for 15 min at 10 C) in a sterile Falcon tubes. Then, the pellet containing bacterial cells and other debris was washed with 20 ml of sterile 0.9% sodium chloride solution, and centrifuged as before. Washed pellet was transferred to a sterile 2 ml tube with 25-30 mg of acid-washed glass beads (212-300 m) (Sigma-Aldrich). Afterwards, extraction was carried out in 180 l ATL buffer (from a kit) in FastPrep-24 5G Instrument (MP Biomedicals, St. Ana, CA, USA). The samples were agitated twice for 25 sec with a 5 sec interval, therewith immediately chilled on ice. Subsequently 25 l Proteinase K was added and incubated 30 min at 56 C, therewith 200 l AL buffer was added. From this point, the next steps proceeded acoording to the kits original protocol to obtain 100 l of the final product. The DNA quality was determined using NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific, Wilmington, USA) and gel electrophoresis. All DNA samples were quantified using Qubit 2.0 Fluorometer with Qubit DNA HS Assay Kit (Thermo Fisher Scientific). Whole DNA was used for microbiota profiling using Oxford Nanopore technology (ONT). The library was prepared following strictly the ONT protocol 16S Barcoding Kit 1-24 (SQK-16S024) (16S_9086_v1_revH_14Aug2019) with recommended third party consumables.