RNA-Seq of Knufia petricola: at pH9
MEB (20 ml) in Erlenmeyer flasks was inoculated with dispersed cells from 8-d-old biofilm cultures of K. petricola A95 (wild type) and incubated for 3 d at 25 C and 100 rpm. Cells were harvested by centrifugation, washed twice with BG11/MEB (10:1, pH 5) and re-suspended in BG11/MEB (10:1) (20 ml) with pH 9. Cultures were incubated for 2 h at 25 C. Cells were lysed in TRI Reagent with glass beads for 2 x 30 s at 5.5 Hz in a Hybaid RiboLyser. After centrifugation, the supernatant was extracted with chloroform. RNA was precipitated from the aqueous phase with 1 volume of isopropanol. Finally, the RNA pellet was washed twice with 75 % ethanol, dried and dissolved in nuclease-free water. 10 g of RNA was incubated with DNaseI and then purified using the RNeasy Kit. Six replicates were prepared and pooled into two samples after RNA extraction. Libraries for RNA-Seq were prepared by Eurofins Genomics (Ebersberg, Germany) using the Eurofins 3-fragment protocol in which total RNA is fragmented, poly-A enriched and cDNA is created by oligo dT priming. Each sample was individually barcoded and the pooled library was sequenced on a Illumina HiSeq 2000 at Eurofins Genomics with chemistry v3.0 and with 100 bp single-end reads.