title: GSM8577157: Triple species biofilmes_NYC III, biol rep 3; Gardnerella vaginalis; Prevotella bivia; Fannyhessea vaginae; RNA-Seq
library_construction_protocol: Twelve triple-species biofilms formed in either NYCIII or mGTS were pooled to obtain samples with enough RNA concentration for further analysis. RNA extraction was performed using the RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions, with some brief modifications as optimized before (França et al., 2012). First, cells were suspended in 600 μL of lysis buffer RLT and the volume was transferred to a tube with 0.1 mm zirconium beads (Merck, Darmstadt, Germany). Cells were lysed using the BeadBug 6 Microtube Homogenizer (Benchmark Scientific, NJ, USA) at maximum speed for 35 s. The cycle was repeated four times and the samples were kept on ice for 5 min between cycles. Then, the samples were centrifuged, and the supernatant was recovered into a new tube. Ethanol at 70% (Thermo Fisher Scientific) was added in the same proportion (vol:vol) to the supernatant and the solution was transferred to an RNeasy Mini spin column. After the washing steps in the column, the RNA was eluted in RNase-free water (Grisp). RNA was treated with Turbo DNase (Invitrogen, Waltham, Massachusetts, USA) to degrade genomic DNA following the manufacturer's instructions for rigorous protocol. RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent, CA, USA) and only samples with RNA quality indicators above 7 were used. RNA-seq libraries were prepared by Lexogen (outsourced) using Lexogen's CORALLTM Total RNA-seq kit with 100 ng of total RNA. Before RNA-seq, rRNA was removed using the RiboCop for Bacteria (Mixed Bacterial Samples META) rRNA Depletion kit (Lexogen). Sequencing libraries were evaluated for quality on a Fragment Analyzer System (Agilent) and quantified with QubitTM dsDNA HS Assay Kit (Invitrogen). Data were generated using Illumina NextSeq2000 Sequencing from single-end reads (SR100).