HiSeq X Five paired end sequencing; RNA-seq of PBT2-Zn treated Fusobacterium nucleatum cells in vitro
To investigate the effect of PBT2 on the transcriptomic response of F. nucleatum, RNA was extracted from bacterial samples which had been treated as follows: Untreated (0.006% DMSO v/v), DMSO-Zn (0.006% DMSO v/v + 200uM ZnSO4), or PBT2-Zn treated (0.125ug/mL PBT2 + 200uM ZnSO4). Samples (30mL) were collected at 0h, 0.5h and 1h post challenge. Samples were transferred into pre-cooled centrifuge tubes containing 60mL ice cold 3:2 glycerol:saline, gently mixed, and centrifuged at 24,000 xg, 20 min, at -10C. The glycerol saline supernatant was discarded and cell pellets were resuspended on ice in 1mL 1:1 glycerol:saline before transfer to sterile eppendorf tubes. Cell pellets were snap frozen in dry ice ethanol bath and stored at -80C until RNA extraction. Cell pellets were resuspended in 1mL TRIzol and disrupted by bead beating three times at 4800 rpm, 30 secs. Tubes were cooled on ice between beating for 30 secs. Tubes were centrifuged at 17,000 xg, 30 secs, at 4C to separate cell debris and beads from intracellular material. Supernatants were transferred to eppendorf tubes. RNA was extracted following the phase separation method by the addition of 200uL chloroform to TRIzol treated cells and centrifugation at 17,000 xg, 15 min, at 4C. The aqueous phase was combined with equal parts absolute ethanol and RNA was extracted using an RNA clean and concentrator kit (ZYMO research). RNA was eluted in 15uL RNase-free water and RNA quality was measured using a NanoDrop. Total RNA was DNase treated using a TURBO DNA-free kit. The library was prepared using a Ribo-Zero stranded protocol (250-300 bp insert strand specific library with rRNA removal using Ribo-Zero Magnetic kit).