GSM8285958: Gardnerella vaginalis single biofilms_biol rep2 (br2); Gardnerella vaginalis; RNA-Seq
RNA extraction was performed using the RNeasy Mini Kit (QIAGEN), according to the manufacturer's instructions with some brief modifications. First, cells were suspended in 600 μL of the lysis buffer RLT and the volume was transferred into a tube with 0.1 mm zirconium beads (Merck). Cells were lysed using the BeadBug 6 Microtube Homogenizer (Benchmark Scientific) at maximum speed for 35s. 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 [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. RNA was then eluted in RNase-free water (GRISP) and treated with Turbo DNase (Invitrogen) to degrade genomic DNA following the manufacturer's instructions for rigorous protocol. RNA quality was assessed using an Agilent 2100 Fragment Analyzer system (Agilent) 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).