GSM5699204: Free-living cells with maltose rep 3; Zobellia galactanivorans; RNA-Seq
Free-living bacterial cells were lysed by adding 400 μl of lysis buffer (4 M guanidine thiocyanate, 25 mM sodium acetate pH 5.2, 5 g.l-1 N-laurylsarcosinate) and 500 μl of phenol and incubated 5 min at 65 °C. RNA was extracted using phenol-chloroform. Extracts were treated 1 h at 37 °C with 2 units of Turbo DNAse and purified on mini-columns NucleoSpin RNA Clean-up following the manufacturer's instructions. RNA was eluted in 50 μl of nuclease-free water. RNA from algae-attached bacteria was extracted as follows. Two algal pieces were immersed in killing buffer, vortexed and placed 7 min in an ultrasonic bath to detach bacteria from the algal surface. Algae were removed and cell pellets resuspended in lysis buffer. RNA extraction and DNAse treatment were performed as described above for the free-living bacteria. DNAse were inactivated using DNAse inactivation reagent following the manufacturer instructions. Librairies were performed by the Plateforme de Séquençage I2BC (UMR9198, CNRS, Gif-sur-Yvette) on a NextSeq instrument (Illumina) using the NextSeq 500/550 High Output Kit v2 (75 cycles) which included a Ribo-Zero ribosomal RNA depletion step.