GSM2113322: Planktonic; Gardnerella vaginalis AMD; RNA-Seq
Biofilm or planktonic cells total RNA was extracted using RNeasy mini kit (QIAGEN) with minor alterations as described in França et al., 2012, PLoS One 7 (5): e37480. In brief, bacterial cells were suspended in 500 µL of in the lysis buffer provided by the kit plus 500 µL of phenol. This suspension was transferred into BeadBug tubes (1.0 mm Zirconium beads, triple-pure, high impact, Sigma) and using a MPBio 24 (MPBio) the cells were lysed. Finally, the tubes were centrifuged and the lysate transferred into a new tube. An equal volume of 70% of ethanol was added. The suspension was transferred in to the RNeasy mini kit columns and the manufacturer's instructions were strickly followed. Total RNA, eluted in 50 µL, was treated with TURBO DNase (Ambion) and 1:5 Acid-phenol:chloroform in order to degrade and isolate, respectively, genomic DNA. Additionally, total RNA was treated with Ribo-Zero rRNA removal kit for Gram-positive bacteria (Epicentre) to remove bacterial ribosomal RNA. Libraries were constructed using ScriptSeq RNAseq Library preparation kit (low input) and strictly following the manufacturer's indications.
GEO Accession
GSM2113322